Compositions and methods for modulating hair growth

ABSTRACT

The present disclosure relates to compounds that are capable of inhibiting the mitochondrial pyruvate carrier and promoting hair growth. The disclosure further relates to methods of promoting hair growth or treating conditions or disorders affecting hair growth, such as baldness or alopecia.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/787,609, filed Jan. 2, 2019, the contents of which are fullyincorporated by reference herein.

BACKGROUND

Hair follicle stem cells (HFSCs) undergo successive rounds of quiescence(telogen) punctuated by brief periods of proliferation correlating withthe start of the hair cycle (telogen-anagen transition). Proliferationor activation of HFSCs is well known to be a prerequisite foradvancement of the hair cycle. Despite advances in treatment options,baldness and alopecia continue to be conditions that cannot besuccessfully treated in all individuals. Some of the existing treatmentsare inconvenient for users, others require surgical intervention orother invasive procedures. Additional therapies are needed.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides compounds of formulaI or a pharmaceutically acceptable salt thereof:

wherein:Y is carboxyl, ester, amide, or

R¹ is H, aryl, aralkyl, or aralkylacyl, and is optionally substituted byone or more R⁵;R² is CN or carboxyl;R⁴ is independently alkyl, alkenyl, alkynyl, carboxyl, azido, halo,hydroxy, ester, or CN;R⁵ is independently selected from alkyl, alkoxy, or halo; andn is 0-4.

In certain embodiments, the present disclosure provides compounds offormula Ia or a pharmaceutically acceptable salt thereof:

wherein R⁶ is H, alkyl, aryl, or aralkyl,

In certain aspects, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present disclosure and apharmaceutically acceptable excipient.

In certain aspects, the present disclosure provides methods of enhancinglactate production in a cell, comprising contacting the cell with acompound or composition of the disclosure.

In certain aspects, the present disclosure provides methods ofinhibiting mitochondrial pyruvate oxidation in a cell, comprisingcontacting the cell with a mitochondrial pyruvate oxidation (MPO)inhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor. In certain embodiments, inhibiting mitochondrial pyruvateoxidation in a cell has the effect of enhancing lactate production in acell and/or enhancing the activity of LDH in a cell, and promoting hairgrowth, as described herein.

In certain aspects, the present disclosure provides methods of enhancinglactate production in a cell, comprising contacting the cell with an MPOinhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor.

In certain aspects, the present disclosure provides methods of enhancingthe activity of LDH in a cell, comprising contacting the cell with anMPO inhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor.

In certain aspects, the present disclosure provides methods of enhancingthe activity of lactic acid dehydrogenase (LDH) in a cell, comprisingcontacting the cell with an MPO inhibitor, such as a compound of thepresent disclosure. In certain embodiments, the MPO inhibitor is amitochondrial pyruvate carrier (MPC) inhibitor. In certain aspects, thepresent disclosure provides methods of promoting hair growth or treatinga hair growth condition or disorder such as baldness or alopecia,comprising administering to a patient a compound or composition asdisclosed herein.

In certain aspects, the present disclosure provides methods of promotinghair growth or treating a hair growth condition or disorder such asbaldness or alopecia, comprising administering to a patient an MPOinhibitor (e.g., topically, such as with a pharmaceutical compositionformulated for topical application), such as a compound of the presentdisclosure. In certain embodiments, the present disclosure providesmethods of promoting hair growth or treating a hair growth condition ordisorder such as baldness or alopecia, comprising administering to apatient an MPC inhibitor (e.g., topically, such as with a pharmaceuticalcomposition formulated for topical application), such a compound of thepresent disclosure. In certain embodiments, inhibiting mitochondrialpyruvate oxidation or the mitochondrial pyruvate carrier in a cell hasthe effect of enhancing lactate production and/or enhancing the activityof LDH in a cell, and promoting hair growth, as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show Lactate dehydrogenase activity is enriched in HFSCs.FIG. 1A: IHC staining for Ldha expression across the hair cycle showsLdha protein confined to the HFSC niche, the bulge, indicated by thebracket. IHC staining for Sox9 on serial sections demarcates the HFSCpopulation. Scale bar indicates 20 micrometers. FIG. 1B: Immunoblottingon FACS-isolated HFSC populations (α6low/Cd34+ and α6hiCd34+) versustotal epidermis (Epi) shows differential expression of Ldha in the stemcell niche. Sox9 is a marker of HFSCs, and β-actin is a loading control.FIG. 1C: Colorimetric assay for Ldh enzyme activity in the epidermisshows highest activity in the bulge (brackets) and subcuticular musclelayer (bracket). This activity is enriched in the bulge across differentstages of the hair cycle. Activity is indicated by purple color; pink isa nuclear counterstain. Note also that developing hair shafts inpigmented mice show strong deposits of melanin as observed here; hairshafts never displayed any purple stain indicative of Ldh activity.Scale bars indicate 50 micrometers. FIG. 1D: Ldh activity in sorted cellpopulations, measured using a plate reader-based assay, also shows thehighest Ldh activity in two separate HFSC populations (α6hi/Cd34 andα6low/Cd34) compared to epidermal cells (Epi) and fibroblasts (FBs).Each bar represents the average signal for each cell type where n=9 micepooled from 3 independent experiments. Shown as mean±SEM. Paired t-testwas performed, p<0.05 shown for each cell type versus epidermal cells.FIG. 1E: HFSCs and epidermal cells were isolated during telogen (day 50)by FACS, and metabolites were extracted and analyzed by LC-MS. Heat mapsshow relative levels of glycolytic and TCA cycle metabolites from cellsisolated from different mice in independent experiments with cells fromthree animals in each. Asterisks indicate significant difference inmetabolite levels between epidermal cells and HFSCs. For e, pairedt-test was performed; *denotes p<0.05, **denotes p<0.01, ***denotesp<0.001, ns denotes p>0.05, and n=9 mice pooled from 3 independentexperiments.

FIGS. 2A-2C show the validation of key reagents and assays. FIG. 2A: IHCwith antibody recognizing specifically Ldha (same as used in FIG. 1A).bottom, IHC with antibody recognizing multiple isoforms of Ldh protein.Scale bars indicate 20 micrometers. FIG. 2B: the sorting strategyemployed to isolate two populations of cells from the bulge. Thisparticular sort was used to isolate the protein samples shown by westernblot in FIG. 1B. FIG. 2C: Validation of colorimetric Ldh enzyme activityassay. The highest Ldh enzyme activity was observed in HFSC bulge and inthe muscle. Activity indicated by purple stain; pink color is nuclearfast red counterstain. In absence of substrate lactate there was nodetectable activity (purple stain). right, Additional validation ofcolorimetric Ldh enzyme activity assay. Enzyme activity inhibited bytreating skin with HCl before addition of staining solution withsubstrate lactate. No Ldh activity (purple stain) detected. Skin inwhich enzyme activity is not inhibited by Hydrochloric Acid (HCl) showshighest Ldh enzyme activity in HFSC bulge and in the muscle. Scale barsindicate 50 micrometers.

FIGS. 3A-3E show that Ldh activity increases during HFSC activation.FIG. 3A: GSEA on RNA-seq transcriptome data from HFSCs versus totalepidermis shows enrichment for Glycolysis related genes in HFSCs(NES=1.72). FIG. 3B: GSEA on microarray transcriptome data from HFSCsversus total epidermis shows enrichment for Glycolysis related genes inHFSCs (NES=1.45). Results were generated from three mice of eachcondition. FIG. 3C: RNA-seq data from HFSCs sorted during telogen ortelogen-anagen transition show induction of Ldha²¹. Data represent theaverage of three separate animals at each timepoint. FIG. 3D: Ldhactivity in sorted stem cell populations, measured using a platereader-based assay, shows elevated Ldh activity as stem cells becomeactivated in telogen to anagen transition (Tel-Ana). Each bar representsthe average signal for each condition where n=9 mice pooled from 3independent experiments. Shown as mean±SEM. Paired t-test was performed,p<0.05. FIG. 3E: Heatmap showing relative levels of glycolytic and TCAcycle metabolites extracted from quiescent (Telogen, day 50), activated(Telogen-Anagen, day 70) and HFSCs that have returned to the quiescentstate (Anagen, day 90). Data shown were generated from n=3 animals pertime point in 3 independent experiments.

FIGS. 4A-4B show validation of hair cycle stage measurements. FIG. 4A:Analysis of RNA-seq data to validate that HFSCs in telogen-anagentransition were in fact in such a transition. The telogen-anagentransition is known to be driven by Shh (Gli factors are targets) andWnt (Lef1, Axin, Ccnd1 are targets) signaling, and correlate withincreased proliferation (Ki67 and Pcna). In addition, Sox4 waspreviously identified as a regulator of the telogen-anagen transition.n=3 mice per timepoint. Shown as mean±SEM. Paired t-test was performed,p<0.05. FIG. 4B: staining for Ki-67 marks dividing cells during variousstages of the hair cycle. Brackets indicate the HFSC niche. Scale barsindicate 100 micrometers.

FIGS. 5A-5G show that deletion of Mpc1 increases lactate production andactivation of HFSCs. FIG. 5A: Mpc1 fl/fl animals show pigmentation andhair growth, consistent with entry into the anagen cycle at 8.5 weeks,whereas Mpc1+/+ animals do not show dorsal pigmentation and hair growththis early. Animals shown are representative of at least 12 animals ofeach genotype. FIG. 5B: FACS isolation of HFSC bulge populations inMpc1+/+ versus Mpc1 fl/fl mice followed by western blotting showssuccessful deletion of Mpc1 protein in the stem cell niche. β-actin is aloading control. FIG. 5C: Plate reader assay for Ldh activity on sortedHFSC populations shows elevated activity in Mpclfl/fl HFSCs compared toMpc1+/+ HFSCs. Each bar represents the average signal for each genotypewhere n=9 mice pooled from 3 independent experiments. Shown as mean±SEM.Paired t-test was performed, p<0.05. FIG. 5D: Histology on WT versusMpc1 deletion skin shows induction of anagen in absence of Mpc1. Scalebars indicate 100 micrometers. Quantification of phenotype at rightshows percentage of dorsal follicles in telogen, telogen to anagentransition and anagen in Mpc1+/+ mice versus Mpc1 fl/fl mice (n=250follicles from 3 mice per genotype). Shown as mean±SEM. Paired t-testwas performed, p<0.05. FIG. 5E: Immunohistochemistry staining for Ki-67,a marker of proliferation that is only active in HFSCs at the beginningof a new hair cycle, is only present in Mpc1fl/fl HFSCs at 8.5 weeks,consistent with their accelerated entry into a new hair cycle.Phospo-S6, another marker that is only active in HFSCs at the beginningof a new hair cycle, is only present in Mpc1 fl/fl HFSCs. Staining forSox9 shows that HFSCs are present in Mpc1 deleted niche. Images taken at60× magnification. FIG. 5F: Deletion of Mpc1 in mice bearing theLgr5CreER allele shows strong induction of the hair cycle. Note that redboxes indicate areas of new hair growth. Results are representative ofat least 9 animals per genotype. FIG. 5G: Quantification of pigmentationin the indicated genotypes across three independent litters (n=5 miceper genotype).

FIGS. 6A-6D show the effects of long term deletion of Mpc1 in HFSCs.FIG. 6A: Six months after initiation of deletion of Mpc1 in HFSCs(K15CrePR;Mpc1fl/fl), mice lacking Mpc1 show no deleterious effects asmeasured by the hair cycle (left), pathology (middle, H and E), orstaining for HFSCs (right, Sox9). Scale bars indicate 100 micrometers inmiddle panel, and 50 micrometers in right panel. Images arerepresentative of at least 12 animals per genotype. FIG. 6B: Todemonstrate that the deletion of Mpc1 promotes proliferationspecifically in HFSCs, we used K15CrePR; Ldha^(fl/fl) mice bearing alox-stop-lox-Tomato allele to look at K15+ HFSCs and proliferation withand without Mpc1 deletion (left). In addition, we took advantage of theires-GFP within the Lgr5CreER allele to stain for Ki-67 and GFP and lookfor co-localization with and without Mpc1 deletion (right). Whitebrackets denote bulge area. Scale bars represent 20 micrometers. FIG.6C: Deletion of Mpc1 in mice bearing the Lgr6CreER allele shows nopremature induction of the hair cycle. FIG. 6D: Ldh activity assay onsorted HFSCs from either control or Lgr6CreER mediated Mpc1 deletionmice showed increased activity in cells lacking Mpc1. n=6 mice pergenotype pooled from 2 independent experiments. Shown as mean±SEM.Paired t-test was performed, p<0.05.

FIGS. 7A-7D show that pharmacological inhibition of Mpc1 promotes HFSCactivation. FIG. 7A: Animals treated topically with UK-5099 (20 μM) showpigmentation and hair growth, indicative of entry into anagen, after 8days of treatment. Full anagen, indicated by full coat of hair, isachieved after 14 days of treatment. Mice treated topically with vehiclecontrol do not show pigmentation nor hair growth even after 12 days oftreatment. right, Skin pathology showing that UK-5099 animals enter anaccelerated anagen at 8 weeks typified by down growth of the follicleand hypodermal thickening, while vehicle control treated animals showedneither and remained in telogen. Images shown are representative of atleast 14 mice from 7 independent experiments. Scale bars indicate 100micrometers. FIG. 7B: Graph showing time to observed phenotype invehicle versus UK-5099 treated mice. n=6 mice per condition. Shown asmean±SEM. FIG. 7C: Ldh enzyme activity assay in the epidermis showsstrong activity in HFSCs in vehicle control and UK-5099 treated animals.Ldh enzyme activity also seen in interfollicular epidermis of UK-5099treated animals. Ldh activity is indicated by purple stain; pink isnuclear fast red counterstain. Scale bars indicate 50 micrometers. FIG.7D: Metabolomic analysis of Lactate on HFSCs isolated from UK-5099treated skin for 48 hours; Each bar represents the average signal foreach condition where n=9 mice pooled from 3 independent experiments.Shown as mean±SEM. Paired t-test was performed, p<0.05.

FIG. 8 shows the effect on lactate production of certain Mpc1 inhibitorsdescribed herein.

FIG. 9 shows the effect on lactate production of the certain Mpc1inhibitor described herein.

FIG. 10 shows the EC50 calculation for UK5099 and JXL020.

FIG. 11 shows that the Mpc1 inhibitors of the present invention inducehair growth.

FIG. 12 shows the effect on lactate production of certain Mpc1inhibitors described herein.

FIG. 13 shows the effect on total cell count of certain Mpc1 inhibitorsdescribed herein, normalized to DMSO treatment.

FIG. 14 shows the effect on cell lactate production of certain Mpc1inhibitors described herein, normalized to DMSO treatment.

FIG. 15 shows the effect on total cell count of certain Mpc1 inhibitorsdescribed herein, normalized to DMSO treatment.

FIG. 16 shows the effect on cell lactate production of certain Mpc1inhibitors described herein, normalized to DMSO treatment.

FIG. 17 shows the effect on total cell count of certain Mpc1 inhibitorsdescribed herein, normalized to DMSO treatment.

FIG. 18 shows the effect on cell lactate production of certain Mpc1inhibitors described herein, normalized to DMSO treatment.

FIG. 19 shows the effect on total cell count of certain Mpc1 inhibitorsdescribed herein, normalized to DMSO treatment.

FIG. 20 shows the role of MPC in the oxidation of pyruvate to acetylcoenzyme A.

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the present disclosure provides compounds of formulaI:

wherein:Y is carboxyl, ester, amide, or

R¹ is H, aryl, aralkyl, or aralkylacyl, and is optionally substituted byone or more R⁵;R² is CN or carboxyl;R⁴ is independently alkyl, alkenyl, alkynyl, azido, halo, hydroxy,carboxyl, ester, or CN;R⁵ is independently selected from alkyl, alkoxy, or halo; andn is 0-4.

In certain embodiments of formula I, the compound is not

In certain embodiments of formula I, Y

In certain such embodiments, R¹⁰ is H. In other such embodiments, R¹⁰ isalkyl (e.g., methyl, ethyl, propyl).

In other, preferred, embodiments of formula I, Y is ester or carboxyl.

In certain preferred embodiments of formula I, R² is CN. In otherembodiments, R² is carboxyl.

In certain embodiments of formula I, R¹ is H.

In other, preferred, embodiments of formula I, R¹ is aralkyl (e.g.,benzyl or phenethyl). In certain such embodiments, the aralkyl (e.g.,benzyl or phenethyl) is substituted by one or more R⁵ (preferably on thephenyl ring). In yet other embodiments, R¹ is aralkylacyl (e.g.,phenylacetyl), and is substituted by one or more R⁵ (preferably on thephenyl ring).

In certain embodiments of formula I, R¹ is substituted by one or two R⁵,and wherein each R⁵ is independently selected from fluoroalkyl orfluoro. In certain preferred embodiments, R¹ is substituted by two R⁵,and wherein each R⁵ is trifluoromethyl.

In certain embodiments of formula I, R⁴ is an electron withdrawinggroup. In certain embodiments, R⁴ is selected from iodo, fluoro, alkenyl(e.g., vinyl), CN, azido, alkynyl (e.g., acetylenyl), fluoroalkyl (e.g.,trifluoromethyl), carboxyl, and ester (e.g., methyl ester or ethylester). In certain preferred embodiments, R⁴ is fluoro. In otherpreferred embodiments, R⁴ is ester (e.g., methyl ester or ethyl ester).

In certain embodiments, the present disclosure provides compounds offormula Ia or a pharmaceutically acceptable salt thereof:

wherein R⁶ is H, alkyl, aryl, or aralkyl.

In certain preferred embodiments of formula Ia, R⁶ is H or alkyl (e.g.,methyl or ethyl).

In certain aspects, the compound of the disclosure is a compound ofTable 1. In certain embodiments, the compounds of Table 1 are MPOinhibitors, for example, the compounds of Table 1 can inhibit MPC1.

TABLE 1 Exemplary Compounds

Additional exemplary compounds are listed in Table 2. In certainembodiments, the compounds of Table 1 are MPO inhibitors, for example,the compounds of Table 1 can inhibit MPC1. In certain embodiments, thecompounds of the present disclosure are not selected from those listedin Table 2.

TABLE 2 Exemplary Compounds

JXL002

JXL003

JXL004

JXL005

JXL006

JXL007

JXL008

JXL009

JXL010

JXL011

JXL012

JXL013

JXL014

JXL015

JXL016

JXL017

JXL018

JXL019

JXL020

JXL021

JXL022

JXL023

JXL024

JXL025

JXL026

JXL027

JXL028

JXL029

JXL030

JXL031

JXL032

JXL033

JXL034

JXL035

JXL036

JXL037

JXL038

JXL039

JXL040

JXL041

JXL042

JXL043

JXL044

JXL045

JXL046

JXL047

JXL048

JXL049

JXL050

JXL051

JXL052

JXL053

JXL054

JXL055

JXL056

JXL057

JXL058

JXL059

JXL060

JXL061

JXL062

JXL063

JXL064

JXL065

JXL066

JXL067

JXL068

JXL069

JXL070

JXL071

JXL072

JXL073

JXL074

JXL075

JXL076

JXL077

JXL078

JXL079

JXL080

JXL081

JXL082

JXL083

JXL084

JXL085

JXL086

JXL087

JXL088

JXL089

JXL090

JXL091

JXL092

JXL093

JXL094

JXL095

JXL096

In certain aspects, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present disclosure and apharmaceutically acceptable excipient.

In certain aspects, the present disclosure provides methods of enhancinglactate production in a cell, comprising contacting the cell with acompound or composition of the disclosure.

In certain aspects, the present disclosure provides methods ofinhibiting mitochondrial pyruvate oxidation in a cell, comprisingcontacting the cell with a mitochondrial pyruvate oxidation (MPO)inhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor. In certain embodiments, inhibiting mitochondrial pyruvateoxidation in a cell has the effect of enhancing lactate production in acell and/or enhancing the activity of LDH in a cell, and promoting hairgrowth, as described herein.

In certain aspects, the present disclosure provides methods of enhancinglactate production in a cell, comprising contacting the cell with an MPOinhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor.

In certain aspects, the present disclosure provides methods of enhancingthe activity of LDH in a cell, comprising contacting the cell with anMPO inhibitor, such as a compound of the present disclosure. In certainembodiments, the MPO inhibitor is a mitochondrial pyruvate carrier (MPC)inhibitor.

In certain aspects, the present disclosure provides methods of enhancingthe activity of lactic acid dehydrogenase (LDH) in a cell, comprisingcontacting the cell with an MPO inhibitor, such as a compound of thepresent disclosure. In certain embodiments, the MPO inhibitor is amitochondrial pyruvate carrier (MPC) inhibitor. In certain aspects, thepresent disclosure provides methods of promoting hair growth or treatinga hair growth condition or disorder such as baldness or alopecia,comprising administering to a patient a compound or composition asdisclosed herein.

In certain aspects, the present disclosure provides methods of promotinghair growth or treating a hair growth condition or disorder such asbaldness or alopecia, comprising administering to a patient an MPOinhibitor (e.g., topically, such as with a pharmaceutical compositionformulated for topical application), such as a compound of the presentdisclosure. In certain embodiments, the present disclosure providesmethods of promoting hair growth or treating a hair growth condition ordisorder such as baldness or alopecia, comprising administering to apatient an MPC inhibitor (e.g., topically, such as with a pharmaceuticalcomposition formulated for topical application), such a compound of thepresent disclosure. In certain embodiments, inhibiting mitochondrialpyruvate oxidation or the mitochondrial pyruvate carrier in a cell hasthe effect of enhancing lactate production and/or enhancing the activityof LDH in a cell, and promoting hair growth, as described herein.

Discussion

Numerous studies have uncovered unique gene expression signatures inhair follicle stem cells (HFSCs) versus other follicle cells or cells ofthe interfollicular epidermis. Many of these signatures are regulated bytranscription factors that were later shown to play important roles inHFSC homeostasis.

Lactate dehydrogenase is most commonly encoded by the Ldha and Ldhbgenes in mammals, the protein products of which form homo- orhetero-tetramers to catalyze the NADH-dependent reduction of pyruvate tolactate and NAD*-dependent oxidation of lactate to pyruvate. Byimmunostaining, it has been discovered that Ldha is enriched inquiescent HFSCs in situ (telogen) (FIG. 1A), and performingimmunohistochemistry staining (IHC) with an antibody that recognizesboth Ldha and Ldhb showed that only Ldha appears to be localized to theHFSC niche (FIG. 2A).

IHC analysis also showed Ldha expression was enriched in HFSCs (Sox9+)at three stages of the hair cycle (FIG. 1A). Consistently,immunoblotting of lysates from sorted cells showed strong expression ofLdha in the basal HFSCs (α6HiCD34+), and suprabasal (α6LoCD34+) HFSCpopulations relative to total epidermis (FIG. 1B) (Sorting strategy isoutlined in FIG. 2B).

To determine whether Ldha expression patterns correlate with activity ofthe Ldh enzyme, a colorimetric-based enzymatic assay was used to assessLdh activity capacity in situ. Typically performed on protein lysates oraliquots with a plate reader, the Ldh activity assay was adapted to workin situ on frozen tissue sections. Note that since both the in situ andin vitro Ldh activity assays employ use of excess substrate (lactate),the results from these assays reflect the capacity for Ldh activity, andnot the steady-state activity.

Applying this assay to skin samples demonstrated that Ldh activitycapacity was significantly higher in HFSCs, consistent with theexpression pattern of Ldha (FIG. 1C). Furthermore, Ldh activity wasenriched in HFSCs across the hair cycle (FIG. 1C). As a control, assaysconducted without the enzymatic substrate (lactate) or on acid-treatedtissue yielded zero activity (FIG. 2C). To further validate theseresults, we sorted epidermal populations, generated cell lysates on thesorted cells, and performed a similar colorimetric-based enzymatic assayon the sorted cell lysates, which also showed increased Ldh activity inHFSCs (FIG. 1D). To better characterize the metabolism of HFSCs, weperformed metabolomics analysis on sorted populations from mouse skin byliquid chromatography-mass spectrometry (LC-MS) (FIG. 1e ). Severalglycolytic metabolites, including glucose/fructose-6-phosphate,fructose-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate,and lactate, were routinely higher in HFSCs relative to total epidermisacross three independent experiments (isolated from different mice ondifferent days). Conversely, most TCA cycle metabolites were notconsistently different between the epidermis and HFSCs (FIG. 1E).Collectively these results suggest that while all cells in the epidermisuse the TCA cycle extensively to generate energy, HFSCs also haveincreased Ldha expression, Ldh activity, and glycolytic metabolism.

Measuring metabolism across the hair cycle therefore would capture anydynamic changes that occur in HFSCs that correlate with activation orquiescence. Analysis of RNA-seq data from HFSCs isolated during eithertelogen or the telogen-anagen transition demonstrated not only that Ldhais the predominant Ldh isoform expressed in HFSCs (FIG. 3), but is alsoinduced during the telogen-anagen transition (FIGS. 3A and 3B(NIHGEOGSE67404 and GSE51635). To confirm that the cells analyzed byRNA-seq were indeed either in telogen or the telogen to anagentransition, important markers of this transition were assessed includingthe Shh and Wnt pathways (Gli1, 2, 3; Lef1, Axin1, Axin2, Ccnd1) as wellas proliferation markers (Ki-67, Pcna and Sox4) (FIG. 4A).

The in vitro Ldh activity assay on lysates from sorted HFSCs uncovered amodest induction of Ldh activity correlating with the telogen to anagentransition (FIG. 3D). Hair cycle staging was validated by Ki-67immunostaining to determine HFSC activation (FIG. 4B). Additionally,measurements of steady-state metabolites extracted from sorted HFSCsshowed an increase in lactate in HFSCs as they transition from telogento telogen-anagen transition, and then decrease again in anagen as HFSCsreturn to quiescence (FIG. 3E).

To determine whether induction of lactate production could affect HFSCactivation or the hair cycle, we crossed K15CrePR animals to thosefloxed for mitochondrial pyruvate carrier 1 (Mpc1)(K15CrePR;Mpc1^(fl/fl)). Mpc1, as a heterodimer with Mpc2, forms themitochondrial pyruvate carrier MPC, a transporter on the innermitochondrial membrane required for pyruvate entry into themitochondria. Loss of function of Mpc1 has been shown to drive lactateproduction through enhanced conversion of pyruvate to lactate by Ldh.Furthermore, inhibition of MPC results in a decrease in mitochondrialpyruvate oxidation (MPO) to acetyl coenzyme A (FIG. 20).

In animals with Mpc1 deletion in HFSCs, we observed a strongacceleration of the ventral and dorsal hair cycles with all the typicalfeatures of a telogen-anagen transition (FIG. 5A) (n=12 littermatepairs). Mifepristone treated K15CrePR;Mpc1^(fl/fl) animals were the onlyto show any signs of dorsal anagen by day 70. Western blotting on sortedHFSCs validated the loss of Mpc1 protein (FIG. 5B). Importantly,purified HFSCs lacking Mpc1 showed a strong induction of Ldh activity(FIG. 5C). Quantification of the dorsal hair cycle across three pairs oflittermates showed a strong induction of anagen in backskin lacking Mpc1(FIG. 5D, right), and histology showed that the anagen induction wasnormal in appearance with a typical hypodermal expansion (FIG. 5D).Immunostaining demonstrated the induction in Mpc1-null HFSCs of variousmarkers of hair cycle activation such as Ki-67 and pS6, while Sox9expression was unaffected (FIG. 5E). Long term deletion of Mpc1 did notlead to aberrant follicles or exhaustion of HFSCs as judged by pathologyand staining for Sox9 (FIG. 6A). Furthermore, deletion of Mpc1 withLgr5CreER showed a very similar phenotype as deletion with K15CrePR(FIGS. 5F and 5G), validating the fact that deletion of this protein inHFSCs leads to their activation (n=12 pairs of littermates). Finally,immunofluorescence for the Ires-GFP of the Lgr5CreER transgene alongwith Ki-67 and lineage tracing with K15CrePR;Mpc1^(fl/fl); lsl-Tomatomice also demonstrated that the HFSCs were indeed proliferativefollowing induction of Mpc1 deletion by tamoxifen or mifepristone (FIG.6B).

UK-5099 (also designated herein as JXL001) is a well-establishedpharmacological inhibitor of the mitochondrial pyruvate carrier and isknown to promote lactate production as a result in various settings.UK-5099 has the following structure:

Topical treatment of animals in telogen (day 50) with UK-5099 led to arobust acceleration of the hair cycle, as well as minorhyperproliferation of the interfollicular epidermis (FIG. 7A).Quantification of the hair cycle across at least 6 pairs of animals(vehicle vs UK-5099) indicated a strong acceleration of the hair cycle,in as few as 6-9 days (FIG. 7B). Similar to genetic deletion of Mpc1,pharmacological blockade of the mitochondrial pyruvate carrier byUK-5099 for 48 hours during telogen promoted increased Ldh activity inHFSCs and the interfollicular epidermis, consistent with increasedcapacity for lactate production (FIG. 7C). Finally, metabolomic analysisdemonstrated that topical application of UK-5099 increases total levelsof lactate in sorted HFSCs (FIG. 7D).

Compounds were synthesized that could topically promote increasedlactate levels and therefore drive the hair cycle.

The compounds were generally prepared by reaction of the correspondingaldehydes, e.g., for JXL001, 1-phenylindole-3-carboxaldehyde, with ethylcyanoacetate in the presence of 40% aq. L-proline to give exclusivelythe E-isomer of the ethyl 2-cyano-3-(1-phenylindol-3yl)propenoate, e.g.,JXL004. Hydrolysis of the ester with mild lithium hydroxide afforded theE-isomer of the acid, e.g., JXL001. All of the other compounds wereprepared by analogous methods using the specific aldehyde. The twoheterocyclic compounds, JXL023 and JXL024, were prepared from thecondensation of 1-phenyl-indole-3-carboxaldehyde withthiazolidine-2,4-dione and 2-iminothiazolidin-4-one. The structures ofall of the compounds were determined using normal organic chemistrymethods, especially high field proton, carbon, and fluorine NMR spectra.In particular, ³J_(C—H) coupling measurements demonstrated that thecompounds all had the E-stereochemistry about the key carbon-carbondouble bond.

To determine whether these compounds could promote cellular lactateproduction, we treated cultured epithelial cells with the compounds andmeasured lactate levels in the culture media using a Nova BiomedicalBioProfile Basic Analyzer. Briefly, cultured epithelial cells weretreated with DMSO, UK-5099 (also called JXL001), or certain of theexemplary compounds disclosed herein for 24-30 hours, and media lactatelevels were measured and normalized to cell number and duration of theexperiment to acquire a cellular lactate production rate (nmol lactate,million cells, hour). The results are shown in FIGS. 8 and 9.

Lactate production rates of treated cells are shown in FIG. 8. Asexpected since they are UK-5099 analogues, most of the novel compoundsassayed increased lactate production. A separate assay was performed tocalculate the EC₅₀ of some of the compounds as shown in FIG. 10.

To determine the efficacy of the compounds on the hair cycle, mice wereshaved at postnatal day 50, and topically treated with a compounddisclosed herein suspended in lotion in every other day for 3 weeks. Asseen in FIG. 11, all the analogues that showed the ability to promotelactate production in the in vitro assay were also able to stimulatehair growth over the course of 2 weeks.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In preferred embodiments, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as a lotion, cream, or ointment.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a selfemulsifying drug delivery systemor a selfmicroemulsifying drug delivery system. The pharmaceuticalcomposition (preparation) also can be a liposome or other polymermatrix, which can have incorporated therein, for example, a compound ofthe invention. Liposomes, for example, which comprise phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); subcutaneously; transdermally (for example as a patchapplied to the skin); and topically (for example, as a cream, ointmentor spray applied to the skin). The compound may also be formulated forinhalation. In certain embodiments, a compound may be simply dissolvedor suspended in sterile water. Details of appropriate routes ofadministration and compositions suitable for same can be found in, forexample, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231,5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the amount of a compound that is sufficient to elicit the desiredtherapeutic effect. It is generally understood that the effective amountof the compound will vary according to the weight, sex, age, and medicalhistory of the subject. Other factors which influence the effectiveamount may include, but are not limited to, the severity of thepatient's condition, the disorder being treated, the stability of thecompound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

In certain other embodiments, the active compound may be administered ata dosing frequency of less than daily, such as every other day, once perweek or twice per week. In various embodiments, the active compound mayadministered intermittently. For example, the active compound may beadministered once daily or once every other day for a month followed bya month in which the active compound is not administered. Theaforementioned dosing pattern could be repeated in cycles; for example,in a single year the active compound could be administered once daily oronce every other day for six non-sequential (e.g., alternating on/off)months.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans; and other mammals such as equines,cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptablesalts of compounds of the invention in the compositions and methods ofthe present invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts. In certain embodiments, contemplated salts of theinvention include, but are not limited to, 1-hydroxy-2-naphthoic acid,2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaricacid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid,adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid,benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capricacid (decanoic acid), caproic acid (hexanoic acid), caprylic acid(octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, formic acid, fumaric acid, galactaric acid, gentisic acid,d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid,glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid,hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid,lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid,mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid,oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionicacid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid,succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acidsalts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification. See, e.g. “Principles of Neural Science”, McGraw-HillMedical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”,Oxford University Press, Inc. (1995); Lodish et al., “Molecular CellBiology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths etal., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co.,N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”,Sinauer Associates, Inc., Sunderland, Mass. (2000).

Chemistry terms used herein, unless otherwise defined herein, are usedaccording to conventional usage in the art, as exemplified by “TheMcGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill,San Francisco, Calif. (1985).

All of the above, and any other publications, patents and publishedpatent applications referred to in this application are specificallyincorporated by reference herein. In case of conflict, the presentspecification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such asan organic or inorganic compound, a mixture of chemical compounds), abiological macromolecule (such as a nucleic acid, an antibody, includingparts thereof as well as humanized, chimeric and human antibodies andmonoclonal antibodies, a protein or portion thereof, e.g., a peptide, alipid, a carbohydrate), or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Agents include, for example, agents whose structure isknown, and those whose structure is not known. The ability of suchagents to inhibit AR or promote AR degradation may render them suitableas “therapeutic agents” in the methods and compositions of thisdisclosure.

A “patient,” “subject,” or “individual” are used interchangeably andrefer to either a human or a non-human animal. These terms includemammals, such as humans, primates, livestock animals (including bovines,porcines, etc.), companion animals (e.g., canines, felines, etc.) androdents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. As usedherein, and as well understood in the art, “treatment” is an approachfor obtaining beneficial or desired results, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e. not worsening) stateof disease, preventing spread of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agentto a subject will also depend, for example, on the age and/or thephysical condition of the subject and the chemical and biologicalproperties of the compound or agent (e.g., solubility, digestibility,bioavailability, stability and toxicity). In some embodiments, acompound or an agent is administered orally, e.g., to a subject byingestion. In some embodiments, the orally administered compound oragent is in an extended release or slow release formulation, oradministered using a device for such slow or extended release.

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic agents such thatthe second agent is administered while the previously administeredtherapeutic agent is still effective in the body (e.g., the two agentsare simultaneously effective in the patient, which may includesynergistic effects of the two agents). For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially. Thus,an individual who receives such treatment can benefit from a combinedeffect of different therapeutic agents.

A “therapeutically effective amount” or a “therapeutically effectivedose” of a drug or agent is an amount of a drug or an agent that, whenadministered to a subject, will have the intended therapeutic effect.The full therapeutic effect does not necessarily occur by administrationof one dose, and may occur only after administration of a series ofdoses. Thus, a therapeutically effective amount may be administered inone or more administrations. The precise effective amount needed for asubject will depend upon, for example, the subject's size, health andage, and the nature and extent of the condition being treated, such ascancer or MDS. The skilled worker can readily determine the effectiveamount for a given situation by routine experimentation.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto.

Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁₋₃₀ for straight chains, C₃₋₃₀ for branchedchains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both unsubstituted andsubstituted alkyl groups, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone, including haloalkyl groups such as trifluoromethyland 2,2,2-trifluoroethyl, etc.

The term “C_(x-y)” or “C_(x)-C_(y)”, when used in conjunction with achemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, oralkoxy is meant to include groups that contain from x to y carbons inthe chain. C₀alkyl indicates a hydrogen where the group is in a terminalposition, a bond if internal. A C₁-6alkyl group, for example, containsfrom one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen orhydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R⁹, R¹⁰, and R^(1′) each independently represent a hydrogen or ahydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refers to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 10 atoms, more preferably from 5 to 7 atoms.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are consideredto be hydrocarbyl for the purposes of this application, but substituentssuch as acetyl (which has a ═O substituent on the linking carbon) andethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbylgroups include, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group-S(O)—.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹

wherein R⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer toan acid addition salt or a basic addition salt which is suitable for orcompatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compoundsrepresented by formula I or II. Illustrative inorganic acids which formsuitable salts include hydrochloric, hydrobromic, sulfuric andphosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts may exist in either ahydrated, solvated or substantially anhydrous form. In general, the acidaddition salts of compounds of formula I or II are more soluble in waterand various hydrophilic organic solvents, and generally demonstratehigher melting points in comparison to their free base forms. Theselection of the appropriate salt will be known to one skilled in theart. Other non-pharmaceutically acceptable salts, e.g., oxalates, may beused, for example, in the isolation of compounds of formula I or II forlaboratory use, or for subsequent conversion to a pharmaceuticallyacceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compounds represented by formula I or II or any of theirintermediates. Illustrative inorganic bases which form suitable saltsinclude lithium, sodium, potassium, calcium, magnesium, or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic, or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

Many of the compounds useful in the methods and compositions of thisdisclosure have at least one stereogenic center in their structure. Thisstereogenic center may be present in a R or a S configuration, said Rand S notation is used in correspondence with the rules described inPure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates allstereoisomeric forms such as enantiomeric and diastereoisomeric forms ofthe compounds, salts, prodrugs or mixtures thereof (including allpossible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds which contain alkenyl groups may exist asZ (zusammen) or E (entgegen) isomers. In each instance, the disclosureincludes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms,although not explicitly indicated in the formulae described herein, areintended to be included within the scope of the present disclosure.

“Prodrug” or “pharmaceutically acceptable prodrug” refers to a compoundthat is metabolized, for example hydrolyzed or oxidized, in the hostafter administration to form the compound of the present disclosure(e.g., compounds of formula I or II). Typical examples of prodrugsinclude compounds that have biologically labile or cleavable(protecting) groups on a functional moiety of the active compound.Prodrugs include compounds that can be oxidized, reduced, aminated,deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,alkylated, dealkylated, acylated, deacylated, phosphorylated, ordephosphorylated to produce the active compound. Examples of prodrugsusing ester or phosphoramidate as biologically labile or cleavable(protecting) groups are disclosed in U.S. Pat. Nos. 6,875,751,7,585,851, and 7,964,580, the disclosures of which are incorporatedherein by reference. The prodrugs of this disclosure are metabolized toproduce a compound of formula I or II. The present disclosure includeswithin its scope, prodrugs of the compounds described herein.Conventional procedures for the selection and preparation of suitableprodrugs are described, for example, in “Design of Prodrugs” Ed. H.Bundgaard, Elsevier, 1985.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filter, diluent, excipient, solvent or encapsulatingmaterial useful for formulating a drug for medicinal or therapeutic use.

The term “Log of solubility”, “Log S” or “log S” as used herein is usedin the art to quantify the aqueous solubility of a compound. The aqueoussolubility of a compound significantly affects its absorption anddistribution characteristics. A low solubility often goes along with apoor absorption. Log S value is a unit stripped logarithm (base 10) ofthe solubility measured in mol/liter.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Preparation of Exemplary Analogous Compounds

To the solution of indole-3-carboxaldehyde (2.8 mmol, 411 mg) in dry DMF(6 mL) were added Cu₂O (0.3 equiv, 0.84 mmol, 120 mg), K₂CO₃ (2.0 equiv,5.6 mmol, 774 mg), and iodobenzene (2.0 equiv, 5.6 mmol, 624 μL)sequentially. The reaction was stirred and refluxed for 24 h, at whichpoint TLC indicated that the reaction was completed. After it was cooledto 21° C., the reaction mixture was filtrated through a Celite padeluting with ethyl acetate. The filtrate was washed by saturated NaClsolution and organic phase was dried by sodium sulfate and concentrated.The residue was purified by flash column chromatography(hexanes/EtOAc=8:1) to provide the desired product. yield: 89%, 550.7mg.

To the solution of 1-phenyl-indole-3-carbaldehyde (1 mmol, 221 mg) inethanol (1 mL) were added ethyl 2-cyanoacetate (1.3 equiv, 1.3 mmol, 140μL) and L-proline (40 mol %, 0.4 mmol, 58 mg). The reaction was stirredat 21° C. for 12 h and yellow solid precipitated gradually. Aftercompletion of the reaction, ice-cold water (2 mL) was added into thereaction vial. The solid was separated by Buchner funnel filtration andwashed with water (2 mL×3) and dried to afford the desired product.yield: 95%, 300 mg.

To the solution of (E)-ethyl 2-cyano-3-(1-phenyl-1H-indol-3-yl)acrylate(0.32 mmol, 100 mg) in THF (2 mL) was added 0.5N LiOH solution (3 equiv,0.6 mmol, 1.2 mL). The reaction mixture was stirred at 21° C. for 1 h.After reaction completion shown by TLC, THF was evaporated. ConcentratedHCl was added dropwise to acidify the reaction mixture until the pH waslower than 1, meanwhile yellow solid precipitated. Ice-cold water (5 mL)was added to the reaction mixture and the solid was separated by Buchnerfunnel filtration and washed with water (5 mL×3). After dried by vacuum,the solid was washed by 2 mL of solvent mixture (hexanes/EtOAc=5:1) 5 to10 times and monitored by TLC until non-polar impurities disappear (Thenon-polar compound was the retro-Aldol condensation product, which canbe recovered from the filtrate). Finally, the purity of the product waschecked by NMR. yield: 65%, 60 mg.

(E)-2-Cyano-3-(1-phenyl-1H-indol-3-yl)acrylic acid (JXL001/UK5099)

¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.56 (s, 1H), 8.06 (m, 1H),7.65 (m, 4H), 7.53 (m, 2H), 7.34 (m, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 145.6, 137.7, 136.3, 133.6, 130.5,128.9, 128.0, 125.0, 124.9, 123.3, 119.9, 118.4, 111.9, 96.7.

The following compounds were synthesized by a route similar to thatdescribed for JXL001: JXL002, JXL003, JXL004, JXL005, JXL006, JXL007,JXL012, JXL013, JXL014, JXL021, JXL025, JXL026, JXL027, JXL028, JXL029,JXL035, JXL093.

(E)-2-Cyano-3-(1H-indol-3-yl)acrylic acid (JXL002)

¹H NMR (500 MHz, DMSO-d₆) δ 12.48 (s, 1H), 8.51 (s, 1H), 8.49 (s, 1H),7.91 (d, J=6.5 Hz, 1H), 7.53 (d, J=7.0 Hz, 1H), 7.23 (m, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.0, 146.5, 136.5, 132.4, 127.3, 123.9,122.4, 118.9, 118.8, 113.2, 110.2, 94.0.

Ethyl (E)-2-cyano-3-(1H-indol-3-yl)acrylate

¹H NMR (500 MHz, CDCl₃) δ 12.55 (s, 1H), 8.53 (s, 1H), 8.52 (s, 1H),7.92 (d, J=7.6 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.26 (app. t, J=7.4 Hz,1H), 7.22 (app. t, J=7.4 Hz, 1H), 4.24 (q, J=7.0 Hz, 2H), 1.26 (t, J=7.0Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.6, 147.0, 136.6, 133.0, 127.3, 124.0,122.5, 118.9, 118.4, 113.3, 110.3, 92.6, 61.8, 14.5.

Ethyl (E)-2-cyano-3-(1-phenyl-1H-indol-3-yl)acrylate (JXL004)

¹H NMR (500 MHz, CDCl₃) δ 8.71 (s, 1H), 8.66 (s, 1H), 7.90 (d, J=7.2 Hz,1H), 7.54 (m, 6H), 7.36 (m, 2H), 4.39 (q, J=7.1 Hz, 2H), 1.42 (t, J=7.1Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.6, 145.6, 137.8, 136.4, 133.2, 129.9,128.5, 124.8, 124.4, 123.0, 118.5, 117.9, 111.6, 111.5, 95.4, 62.0,14.3.

Ethyl (E)-3-(6-chloro-1-phenyl-1H-indol-3-yl)-2-cyanoacrylate (JXL005)

¹H NMR (500 MHz, CDCl₃) δ 8.67 (s, 1H), 8.58 (s, 1H), 7.81 (d, J=8.5 Hz,1H), 7.60 (m, 2H), 7.52 (m, 4H), 7.34 (d, J=8.4 Hz, 1H), 4.39 (q, J=7.1Hz, 2H), 1.41 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.4, 145.1, 137.3, 136.8, 133.5, 130.5,130.0, 128.8, 126.8, 124.8, 123.6, 119.5, 117.5, 111.6, 111.4, 96.4,62.1, 14.2.

Ethyl (E)-2-cyano-3-(1-(2-methoxyphenyl)-1H-indol-3-yl)acrylate (JXL006)

¹H NMR (500 MHz, CDCl₃) δ 8.67 (s, 1H), 8.66 (s, 1H), 7.89 (d, J=7.8 Hz,1H), 7.49 (app. t, J=8.6 Hz, 1H), 7.41 (d, J=7.6 Hz, 1H), 7.35 (app. t,J=7.3 Hz, 1H), 7.30 (t, J=7.5 Hz, 1H), 7.23 (d, J=8.1 Hz, 1H), 7.13 (m,2H), 4.39 (q, J=7.1 Hz, 2H), 3.81 (s, 3H), 1.41 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.8, 154.2, 146.0, 137.2, 135.2, 130.3,127.8, 126.0, 124.0, 122.6, 120.8, 118.2, 118.0, 112.3, 111.8, 111.0,94.7, 61.8, 55.7, 14.3.

Ethyl (E)-2-cyano-3-(1-(4-methoxyphenyl)-1H-indol-3-yl)acrylate (JXL007)

¹H NMR (500 MHz, CDCl₃) δ 8.65 (s, 1H), 8.64 (s, 1H), 7.89 (d, J=7.2 Hz,1H), 7.44 (m, 3H), 7.35 (m, 2H), 7.07 (d, J=8.8 Hz, 2H), 4.38 (q, J=7.1Hz, 2H), 3.90 (s, 3H), 1.41 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.8, 159.5, 145.6, 136.8, 133.5, 130.6,128.3, 126.2, 124.2, 122.9, 118.4, 118.0, 115.0, 111.5, 111.1, 94.9,61.9, 55.6, 14.2.

(E)-2-Cyano-3-(1-(4-methoxyphenyl)-1H-indol-3-yl)acrylic acid (JXL012)

¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.52 (s, 1H), 8.05 (d, J=7.7Hz, 1H), 7.58 (app. d, J=8.7 Hz, 2H), 7.44 (m, 1H), 7.33 (m, 2H), 7.16(app. d, J=8.7 Hz, 2H), 3.82 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 160.5, 136.7, 133.9, 130.5, 127.8,126.6, 124.8, 123.2, 120.0, 119.5, 118.6, 115.6, 115.4, 111.9, 110.9,55.9.

(E)-3-(6-Chloro-1-phenyl-1H-indol-3-yl)-2-cyanoacrylic acid (JXL013)

¹H NMR (500 MHz, DMSO-d₆) δ 13.58 (br. s, 1H), 8.59 (s, 1H), 8.54 (s,1H), 8.11 (d, J=7.5 Hz, 1H), 7.67 (m, 4H), 7.53 (m, 2H), 7.35 (d, J=7.5Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.3, 145.4, 137.3, 136.7, 134.4, 130.6,129.6, 129.1, 126.7, 125.1, 123.5, 121.5, 118.2, 111.6, 111.3, 97.9.

(E)-2-Cyano-3-(1-(2-methoxyphenyl)-1H-indol-3-yl)acrylic acid (JXL014)

¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.47 (s, 1H), 8.02 (d, J=7.4Hz, 1H), 7.54 (m, 2H), 7.31 (m, 3H), 7.15 (m, 2H), 3.74 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.6, 154.2, 145.8, 137.3, 135.1, 131.1,128.2, 127.3, 125.7, 124.6, 123.0, 121.5, 119.3, 118.5, 113.6, 112.2,110.8, 96.1, 56.2.

2-((1-Phenyl-1H-indol-3-yl)methylene)malononitrile (JXL021)

¹H NMR (500 MHz, CDCl₃) δ 8.62 (s, 1H), 8.13 (s, 1H), 7.80 (d, J=7.4 Hz,1H), 7.61 (m, 2H), 7.53 (m, 4H), 7.40 (m, 2H).

¹³C NMR (126 MHz, CDCl₃) δ 149.8, 137.3, 136.5, 133.6, 130.1, 128.9,127.7, 124.9, 124.8, 123.7, 118.2, 115.1, 115.0, 111.9, 111.8, 73.7.

2-(Ethoxycarbonyl)-3-(1-phenyl-1H-indol-3-yl)acrylic acid (a mixture ofE/Z isomers, 1:1 ratio) (JXL025)

¹H NMR (500 MHz, DMSO-d₆) δ 13.08 (br. s, 1H), 7.87 (m, 3H), 7.61 (m,4H), 7.52 (m, 2H), 7.30 (m, 2H), 4.26 (m, 2H), 1.23 (m, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.9, 167.8, 166.1, 164.9, 138.3, 136.1,132.9, 132.1, 131.1, 130.9, 130.8, 128.5, 124.9, 124.4, 122.6, 122.2,119.5, 111.6, 111.0, 61.7, 61.3, 14.7, 14.4.

(E)-2-Cyano-3-(4-fluoro-1-phenyl-1H-indol-3-yl)acrylic acid (JXL026)

¹H NMR (500 MHz, DMSO-d₆) δ 8.65 (s, 1H), 8.61 (s, 1H), 7.67 (m, 4H),7.57 (m, 1H), 7.36 (m, 2H), 7.19 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.4, 156.8 (d, J_(c-f)=245.6 Hz), 146.4,138.7, 137.6, 133.4, 130.7, 129.5, 125.9, 125.4, 118.2, 116.3, 116.2,109.5 (d, J_(c-f)=34.5 Hz), 109.2 (d, J_(c-f)=23.2 Hz), 98.0.

(E)-2-Cyano-3-(6-fluoro-1-phenyl-1H-indol-3-yl)acrylic acid (JXL027)

¹H NMR (500 MHz, DMSO-d₆) δ 13.59 (br. s, 1H), 8.62 (s, 1H), 8.59 (s,1H), 8.16 (m, 1H), 7.66 (m, 4H), 7.56 (m, 1H), 7.36 (d, J=9.2 Hz, 1H),7.25 m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.6, 160.8 (d, J_(c-f)=240.0 Hz), 145.7,137.6, 136.6, 134.4, 130.8, 129.1, 125.0, 124.7, 121.6, 118.4, 111.8 (d,J_(c-f)=24.2 Hz), 111.5, 98.7 (d, J_(c-f)=26.2 Hz), 97.7.

(E)-2-Cyano-3-(7-fluoro-1-phenyl-1H-indol-3-yl)acrylic acid (JXL028)

¹H NMR (500 MHz, DMSO-d₆) δ 13.62 (br. s, 1H), 8.56 (s, 1H), 8.47 (s,1H), 7.89 (br. s, 1H), 7.61 (m, 5H), 7.30 (br. s, 1H), 7.17 (br. s, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.4, 149.7 (d, J_(c-f)=247.5 Hz), 145.5,139.0, 135.5, 131.9, 129.9, 129.3, 126.2, 124.3, 124.0, 118.3, 115.9,111.8, 110.9 (d, J_(c-f)=17.4 Hz), 98.1.

(E)-2-Cyano-3-(5-fluoro-1-phenyl-1H-indol-3-yl)acrylic acid (JXL029)

¹H NMR (500 MHz, DMSO-d₆) δ 13.57 (br. s, 1H), 8.67 (s, 1H), 8.60 (s,1H), 8.01 (d, J=9.0 Hz, 1H), 7.70 (m, 4H), 7.58 (m, 2H), 7.24 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.6, 159.4 (d, J_(c-f)=237.8 Hz), 145.8,137.7, 135.1, 133.1, 130.7, 129.2, 129.1, 125.2, 118.5, 113.7, 113.2 (d,J_(c-f)=26.5 Hz), 111.4, 105.7 (d, J_(c-f)=24.2 Hz), 97.2.

(E)-3-(4-Chloro-1-phenyl-1H-indol-3-yl)-2-cyanoacrylic acid (JXL035)

¹H NMR (500 MHz, DMSO-d₆) δ 9.22 (s, 1H), 8.68 (s, 1H), 7.64 (m, 3H),7.59 (m, 2H), 7.48 (d, J=7.6 Hz, 1H), 7.38 (m, 1H), 7.32 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 146.4, 138.0, 137.4, 134.5, 130.7,130.5, 129.5, 125.7, 125.3, 124.9, 123.7, 118.4, 111.7, 110.9, 97.5.

Ethyl (E)-3-(4-chloro-1-phenyl-1H-indol-3-yl)-2-cyanoacrylate (JXL093)

¹H NMR (500 MHz, CDCl₃) δ 9.51 (s, 1H), 8.78 (s, 1H), 7.59 (m, 2H), 7.51(m, 3H), 7.40 (dd, J=8.3, 0.8 Hz, 1H), 7.32 (dd, J=7.7, 0.8 Hz, 1H),7.22 (app. t, J=8.0 Hz, 1H), 4.38 (q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz,3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.6, 147.6, 138.1, 137.4, 134.2, 130.1,129.0, 126.9, 125.3, 124.6, 124.5, 124.3, 118.0, 111.8, 110.6, 96.0,62.1, 14.3.

Experimental Details for the Synthesis of JXL020

To the solution of indole-3-carboxaldehyde (3 mmol, 435 mg) in dry DMF(6 mL) were added 3,5-bis(trifluoromethyl)benzyl bromide (1.2 equiv, 3.6mmol, 660 μL) and KOH (1.2 equiv, 3.6 mmol, 200 mg) at 0° C. Thereaction mixture was stirred at 21° C. for 2 h. After the reactioncompletion shown by TLC, water (6 mL) was added to the reaction vial.The reaction mixture was extracted by dichloromethane (15 mL×3). Thecombined organic layer was dried by sodium sulfate and concentrated. Theresidue was purified by flash column chromatography (hexanes/EtOAc=8:1)to provide the desired product. yield: 90%, 1001.7 mg.

To the solution of1-(3,5-bis(trifluoromethyl)benzyl)-1H-indole-3-carbaldehyde (1 mmol, 371mg) in ethanol (1 mL) were added ethyl 2-cyanoacetate (1.3 equiv, 1.3mmol, 140 μL) and L-proline (40 mol %, 0.4 mmol, 58 mg). The reactionwas stirred at 21° C. for 12 h and yellow solid precipitated gradually.After completion of the reaction, ice-cold water (2 mL) was added intothe reaction vial. The solid was separated by Buchner funnel filtrationand washed with water (2 mL×3) and dried to afford the desired product.yield: 93%, 433 mg.

To the solution of (E)-ethyl3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylate(0.21 mmol, 100 mg) in THF (2 mL) was added 0.5N LiOH solution (3 equiv,0.4 mmol, 0.8 mL). The reaction mixture was stirred at 21° C. for 1 h.After reaction completion shown by TLC, THF was evaporated. ConcentratedHCl was added dropwise to acidify the reaction mixture until pH waslower than 1, meanwhile yellow solid precipitated. Ice-cold water (5 mL)was added to the reaction mixture and the solid was separated by Buchnerfunnel filtration and washed with water (5 mL×3). After dried by vacuum,the solid was washed by 2 mL of solvent mixture (hexanes/EtOAc=5:1) 5 to10 times and monitored by TLC until non-polar impurities disappear (Thenon-polar compound was the retro-Aldol condensation product, which canbe recovered from the filtrate). Finally, the purity of the product waschecked by NMR. yield: 55%, 52 mg.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL020)

¹H NMR (500 MHz, DMSO-d₆) δ 13.37 (br. s, 1H), 8.75 (s, 1H), 8.48 (s,1H), 7.99 (m, 4H), 7.65 (s, 1H), 7.28 (m, 2H), 5.83 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 145.7, 140.3, 136.3, 134.8, 131.1,130.8 (q, J=31.1 Hz), 128.9, 128.7, 127.9, 124.8, 124.3, 122.9 (q,J=273.4 Hz), 122.2, 119.3, 118.3, 95.6, 49.2.

The following compounds were synthesized by a route similar to thatdescribed for JXL020: JXL008, JXL009, JXL010, JXL011, JXL015, JXL016,JXL017, JXL018, JXL019, JXL036, JXL037, JXL038, JXL039, JXL040, JXL041,JXL050, JXL051, JXL052, JXL053, JXL054, JXL055, JXL56, JXL057, JXL058,JXL059, JXL060, JXL061, JXL062, JXL063, JXL064, JXL065, JXL066, JXL068,JXL069, JXL072, JXL073, JXL076, JXL077, JXL078, JXL081, JXL082, JXL087,JXL088, JXL089, JXL090, JXL091.

Ethyl (E)-2-cyano-3-(1-(4-fluorobenzyl)-1H-indol-3-yl)acrylate (JXL008)

¹H NMR (500 MHz, CDCl₃) δ 8.60 (app. s, 2H), 7.85 (d, J=6.8 Hz, 1H),7.32 (m, 3H), 7.15 (m, 2H), 7.03 (app. t, 2H), 5.39 (s, 2H), 4.37 (q,J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.7, 162.5 (d, J_(c-f)=247.7 Hz), 145.7,136.1, 133.8, 130.9, 128.6, 128.5, 124.0, 122.7, 118.6, 118.0, 116.0 (d,J_(c-f)=21.9 Hz), 110.9, 110.4, 94.6, 61.9, 50.7, 14.2.

Ethyl (E)-2-cyano-3-(1-(3,4-difluorobenzyl)-1H-indol-3-yl)acrylate(JXL009)

¹H NMR (500 MHz, CDCl₃) δ 8.60 (s, 1H), 8.59 (s, 1H), 7.86 (d, J=7.8 Hz,1H), 7.33 (m, 2H), 7.28 (s, 1H), 7.13 (m, 1H), 6.95 (m, 1H), 6.89 (m,1H), 5.39 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.7, 150.7 (dd, J=251.2, 13.2 Hz), 150.2(dd, J=250.4, 12.6 Hz), 145.7, 136.1, 133.7, 132.3, 128.6, 124.3, 122.9,122.7, 120.0, 118.8, 118.0 (d, J=17.5 Hz), 115.9 (d, J=18.0 Hz), 110.8,110.6, 95.2, 62.1, 50.4, 14.4.

Ethyl (E)-2-cyano-3-(1-(3,5-difluorobenzyl)-1H-indol-3-yl)acrylate(JXL010)

¹H NMR (500 MHz, CDCl₃) δ 8.61 (s, 1H), 8.59 (s, 1H), 7.87 (d, J=7.1 Hz,1H), 7.33 (m, 2H), 7.26 (d, J=7.2 Hz, 1H), 6.75 (app. t, J=8.7 Hz, 1H),6.64 (app. d, J=5.7 Hz, 2H), 5.41 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40(t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.6, 163.3 (dd, J_(c-f)=251.0, 12.5 Hz),145.5, 139.2, 136.0, 133.6, 128.4, 124.3, 122.9, 118.7, 118.0, 110.7,110.6, 109.5 (dd, J_(c-f)=19.9, 6.4 Hz), 103.8 (t, J_(c-f)=25.2 Hz),95.3, 62.0, 50.4, 14.2.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylate(JXL011)

¹H NMR (500 MHz, CDCl₃) δ 8.62 (s, 1H), 8.61 (s, 1H), 7.90 (d, J=7.7 Hz,1H), 7.85 (s, 1H), 7.57 (s, 2H), 7.35 (m, 2H), 7.23 (d, J=7.8 Hz, 1H),5.56 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.6, 145.6, 138.1, 136.0, 133.4, 132.7 (q,J=33.8 Hz), 128.6, 126.8, 124.7, 123.2, 122.9 (q, J=273.4 Hz), 122.6,119.0, 118.0, 111.2, 110.4, 95.9, 62.1, 50.5, 14.3.

(E)-2-Cyano-3-(1-(4-fluorobenzyl)-1H-indol-3-yl)acrylic acid (JXL015)

¹H NMR (500 MHz, DMSO-d₆) δ 8.64 (s, 1H), 8.46 (s, 1H), 7.93 (d, J=7.1Hz, 1H), 7.61 (d, J=7.3 Hz, 1H), 7.33 (m, 2H), 7.26 (m, 2H), 7.16 (m,2H), 5.60 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 162.0 (d, J_(c-f)=244.3 Hz), 145.6,136.4, 134.6, 133.1, 130.0, 128.0, 124.1, 122.8, 119.2, 118.5, 116.0 (d,J_(c-f)=21.7 Hz), 112.0, 109.8, 95.0, 49.6.

(E)-2-Cyano-3-(1-(3,4-difluorobenzyl)-1H-indol-3-yl)acrylic acid(JXL016)

¹H NMR (500 MHz, DMSO-d₆) δ 13.34 (br. s, 1H), 8.62 (s, 1H), 8.47 (s,1H), 7.94 (d, J=7.3 Hz, 1H), 7.61 (d, J=7.6 Hz, 1H), 7.40 (m, 2H), 7.27(m, 2H), 7.10 (br. s, 1H), 5.61 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 149.7 (dd, J_(c-f)=253.3, 13.6 Hz),149.4 (dd, J_(c-f)=246.3, 11.6 Hz), 145.7, 136.4, 134.7, 134.5, 128.0,124.7 (dd, J_(c-f)=5.9, 3.0 Hz), 124.2, 122.8, 119.2, 118.4, 118.3 (d,J_(c-f)=17.0 Hz), 117.1 (d, J_(c-f)=17.6 Hz), 112.0, 109.9, 95.0, 49.3.

(E)-2-Cyano-3-(1-(3,5-difluorobenzyl)-1H-indol-3-yl)acrylic acid(JXL017)

¹H NMR (500 MHz, DMSO-d₆) δ 8.67 (s, 1H), 8.48 (s, 1H), 7.95 (d, J=4.8Hz, 1H), 7.59 (d, J=4.4 Hz, 1H), 7.27 (m, 2H), 7.15 (s, 1H), 6.98 (br.s, 2H), 5.65 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 162.9 (dd, J_(c-f)=247.0, 12.8 Hz),145.6, 141.4, 136.4, 134.8, 128.0, 124.2, 122.9, 119.3, 118.4, 111.9,111.0 (d, J_(c-f)=26.1 Hz), 110.8, 103.8 (t, J_(c-f)=26.5 Hz), 95.5,49.5.

Ethyl (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyanoacrylate (JXL018)

¹H NMR (500 MHz, CDCl₃) δ 8.61 (s, 1H), 8.60 (s, 1H), 7.85 (d, J=7.9 Hz,1H), 7.33 (m, 6H), 7.17 (m, 2H), 5.42 (s, 2H), 4.37 (q, J=7.1 Hz, 2H),1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.8, 145.7, 136.2, 135.1, 134.0, 129.0,128.5, 128.2, 126.8, 124.0, 122.6, 118.5, 118.1, 111.0, 110.3, 94.3,61.8, 51.4, 14.2.

(E)-3-(1-Benzyl-1H-indol-3-yl)-2-cyanoacrylic acid (JXL019)

¹H NMR (500 MHz, DMSO-d₆) δ 13.34 (br. s, 1H), 8.65 (s, 1H), 8.48 (s,1H), 7.93 (d, J=6.9 Hz, 1H), 7.60 (d, J=6.8 Hz, 1H), 7.25 (m, 7H), 5.62(s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 145.7, 136.8, 136.5, 134.7, 129.1,128.2, 127.6, 124.0, 122.7, 120.0, 119.2, 118.5, 112.1, 109.7, 94.7,50.4.

(E)-2-Cyano-3-(1-(3,4-difluorobenzyl)-5-fluoro-1H-indol-3-yl)acrylicacid (JXL036)

¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.47 (s, 1H), 7.84 (d, J=9.6Hz, 1H), 7.63 (dd, J=8.9, 4.3 Hz, 1H), 7.41 (m, 2H), 7.14 (m, 2H), 5.61(s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.9, 159.4 (d, J_(c-f)=237.3 Hz), 149.8(dd, J_(c-f)=247.1, 12.7 Hz), 149.5 (dd, J_(c-f)=246.6, 12.3 Hz), 146.0,136.1, 134.5, 133.1, 129.1, 125.0, 118.5, 118.4, 117.4, 113.6, 112.5 (d,J_(c-f) 26.2 Hz), 110.1, 105.2 (d, J_(c-f)=25.1 Hz), 95.5, 49.7.

(E)-2-Cyano-3-(1-(3,5-difluorobenzyl)-5-fluoro-1H-indol-3-yl)acrylicacid (JXL037)

¹H NMR (500 MHz, DMSO-d₆) δ 8.71 (s, 1H), 8.48 (s, 1H), 7.85 (dd, J=9.6,2.0 Hz, 1H), 7.62 (dd, J=8.9, 4.3 Hz, 1H), 7.16 (m, 2H), 7.00 (app. d,J=6.2 Hz, 2H), 5.65 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.9, 163.0 (d, J_(c-f)=247.7 Hz), 159.3(d, J_(c-f)=237.6 Hz), 145.9, 141.4, 136.3, 133.2, 129.1, 118.5, 113.6,112.6 (d, J_(c-f)=26.3 Hz), 111.3, 110.2, 105.3 (d, J_(c-f)=24.9 Hz),104.0 (t, J_(c-f)=25.2 Hz), 95.9, 49.8.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-5-fluoro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL038)

¹H NMR (500 MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.49 (s, 1H), 8.06 (s, 1H),8.04 (s, 2H), 7.86 (dd, J=9.6, 2.1 Hz, 1H), 7.69 (dd, J=8.9, 4.3 Hz,1H), 7.17 (dt, J=9.0, 2.2 Hz, 1H), 5.83 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 159.3 (d, J_(c-f)=237.5 Hz), 145.9,140.3, 136.2, 133.1, 131.1 (q, J_(c-f)=33.1 Hz), 129.1, 123.6 (q,J_(c-f)=272.2 Hz), 118.4, 113.5, 112.7, 112.5, 110.4, 105.5, 105.3,96.1, 49.6.

(E)-2-Cyano-3-(1-(3,4-difluorobenzyl)-6-fluoro-1H-indol-3-yl)acrylicacid (JXL039)

¹H NMR (500 MHz, DMSO-d₆) δ 8.64 (s, 1H), 8.46 (s, 1H), 7.99 (dd, J=8.7,5.1 Hz, 1H), 7.58 (dd, J=9.8, 1.8 Hz, 1H), 7.43 (m, 2H), 7.12 (m, 2H),5.57 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 160.3 (d, J_(c-f)=239.3 Hz), 149.8(dd, J_(c-f)=239.3, 25.2 Hz), 149.6 (dd, J_(c-f)=246.3, 25.2 Hz), 145.8,136.8, 135.3, 134.5, 125.1, 125.0, 124.6, 121.1, 118.5, 117.5, 111.3 (d,J_(c-f)=23.9 Hz), 110.2, 98.7 (d, J_(c-f)=26.5 Hz), 96.2, 49.4.

(E)-2-Cyano-3-(1-(3,5-difluorobenzyl)-6-fluoro-1H-indol-3-yl)acrylicacid (JXL040)

¹H NMR (500 MHz, DMSO-d₆) δ 8.65 (s, 1H), 8.46 (s, 1H), 8.00 (dd, J=8.6,5.2 Hz, 1H), 7.57 (d, J=9.7 Hz, 1H), 7.15 (m, 2H), 7.03 (s, 1H), 7.02(s, 1H), 5.62 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 163.0 (d, J_(c-f)=239.4 Hz), 162.9(d, J_(c-f)=248.6 Hz), 160.4 (d, J_(c-f)=239.4 Hz), 145.5, 141.3, 136.8,135.2, 124.6, 121.1, 118.4, 111.3, 111.1, 110.3, 104.0 (t, J_(c-f)=25.2Hz), 98.7 (d, J_(c-f)=26.5 Hz), 96.9, 49.6.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-6-fluoro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL041)

¹H NMR (500 MHz, DMSO-d₆) δ 8.74 (s, 1H), 8.48 (s, 1H), 8.06 (app. s,3H), 8.01 (dd, J=8.7, 5.1 Hz, 1H), 7.66 (dd, J=9.8, 2.0 Hz, 1H), 7.13(dt, J=9.3, 2.1 Hz, 1H), 5.78 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 159.9 (d, J_(c-f)=264.6 Hz), 145.8,140.2, 136.8, 135.3, 131.1 (q, J_(c-f)=33.3 Hz), 129.1, 124.5, 123.6 (q,J_(c-f)=273.7 Hz), 122.5, 121.2, 118.4, 111.4 (d, J_(c-f)=25.2 Hz),110.4, 98.6 (d, J_(c-f)=27.2 Hz), 96.7, 49.4.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-chloro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL050)

¹H NMR (500 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.90 (s, 1H), 8.06 (m, 3H),7.72 (d, J=7.5 Hz, 1H), 7.32 (m, 2H), 5.86 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 146.7, 140.0, 138.0, 135.6, 131.1(q, J_(c-f)=33.1 Hz), 129.1, 125.6, 125.1, 124.7, 124.5, 123.7, 123.6(q, J_(c-f)=273.7 Hz), 122.5, 111.6, 109.9, 96.7, 49.6.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-bromo-1H-indol-3-yl)-2-cyanoacrylicacid (JXL051)

¹H NMR (500 MHz, DMSO-d₆) δ 9.37 (s, 1H), 8.91 (s, 1H), 8.07 (app. s,3H), 7.77 (d, J=8.2 Hz, 1H), 7.51 (d, J=7.6 Hz, 1H), 7.23 (t, J=8.0 Hz,1H), 5.85 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 146.3, 140.0, 138.0, 135.8, 131.0(q, J_(c-f)=33.1 Hz), 129.1, 127.9, 125.4, 124.9, 123.7 (q,J_(c-f)=273.2 Hz), 122.5, 118.2, 113.6, 122.1, 110.2, 96.6, 49.5.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-fluoro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL052)

¹H NMR (500 MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.58 (s, 1H), 8.05 (app. s,3H), 7.54 (d, J=8.2 Hz, 1H), 7.29 (d, J=12.9 Hz, 1H), 7.09 (dd, J=11.1,8.2 Hz, 1H), 5.85 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 155.7 (d, J_(c-f)=239.4 Hz), 146.5,140.1, 138.8, 134.5, 131.1 (q, J_(c-f)=33.0 Hz), 129.0, 125.1 (d,J_(c-f)=7.6 Hz), 124.7, 123.6 (q, J_(c-f)=273.5 Hz), 122.5, 118.1, 116.1(d, J_(c-f)=18.5 Hz), 108.8, 108.5, 97.2, 49.7.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-7-fluoro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL053)

¹H NMR (500 MHz, DMSO-d₆) δ 8.73 (br. s, 1H), 8.48 (br. s, 1H), 8.06(br. s, 1H), 7.89 (br. s, 2H), 7.78 (br. s, J=7.4 Hz, 1H), 7.22 (br. s,1H), 7.10 (br. s, 1H), 5.89 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.6, 159.7 (d, J_(c-f)=245.7 Hz), 145.6,141.0, 136.2, 132.1, 130.0 (q, J_(c-f)=33.0 Hz), 128.4, 123.9, 123.8,123.6 (q, J_(c-f)=273.3 Hz), 122.4 (d, J_(c-f)=18.9 Hz), 118.1, 115.8,110.6, 110.2 (d, J_(c-f)=18.9 Hz), 97.3, 52.0.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-5-chloro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL054)

¹H NMR (500 MHz, DMSO-d₆) δ 8.78 (s, 1H), 8.51 (s, 1H), 8.12 (s, 1H),8.06 (s, 1H), 8.03 (s, 2H), 7.70 (d, J=7.4 Hz, 1H), 7.33 (d, J=7.0 Hz,1H), 5.83 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 145.7, 140.2, 135.9, 135.1, 131.1(q, J_(c-f)=33.0 Hz), 129.3, 129.0, 127.9, 124.4, 123.6 (q,J_(c-f)=274.0 Hz), 122.5, 119.5, 118.4, 113.6, 109.9, 96.9, 49.5.

(E)-3-(1-3,5-Bis(trifluoromethyl)benzyl)-4-cyano-1H-indol-3-yl)-2-cyanoacrylicacid (JXL055)

¹H NMR (500 MHz, DMSO-d₆) δ 9.03 (s, 1H), 9.00 (s, 1H), 8.09 (m, 4H),7.80 (d, J=7.3 Hz, 1H), 7.47 (t, J=7.8 Hz, 1H), 5.90 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.4, 144.4, 139.9, 136.7, 131.1 (q,J_(c-f)=33.0 Hz), 129.7, 129.2, 126.6, 124.3, 123.6 (q, J_(c-f)=273.7Hz), 122.7, 118.6, 117.9, 117.8, 109.3, 101.7, 98.1, 49.5.

(E)-1-(3,5-Bis(trifluoromethyl)benzyl)-3-(2-carboxy-2-cyanovinyl)-1H-indole-4-carboxylicacid (JXL056)

¹H NMR (500 MHz, DMSO-d₆) δ 9.26 (br. s, 1H), 8.88 (br. s, 1H), 8.05(app. s, 3H), 7.94 (br. s, 1H), 7.76 (br. s, 1H), 7.37 (br. s, 1H), 5.85(s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 169.3, 165.0, 150.6, 140.2, 137.7, 136.0,131.1 (q, J_(c-f)=33.0 Hz), 129.0, 125.9, 125.2, 124.7, 123.6 (q,J_(c-f)=273.7 Hz), 123.5, 122.5, 118.3, 116.1, 110.1, 96.4, 49.3.

(E)-3-(4-(Benzyloxy)-1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL057)

¹H NMR (500 MHz, DMSO-d₆) δ 9.15 (s, 1H), 8.72 (s, 1H), 8.04 (s, 1H),7.99 (s, 2H), 7.54 (br. s, 3H), 7.37 (br. s, 2H), 7.28 (m, 2H), 6.95 (s,1H), 5.81 (s, 2H), 5.28 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.0, 153.7, 148.6, 140.5, 137.9, 133.4,131.2 (q, J_(c-f)=32.8 Hz), 128.9, 128.8, 128.1, 127.5, 125.4, 124.7,123.6 (q, J_(c-f)=273.7 Hz), 122.3, 118.5, 117.0, 110.5, 105.6, 105.2,95.5, 70.0, 49.5.

(E)-3-(6-(Benzyloxy)-1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL058)

¹H NMR (500 MHz, DMSO-d₆) δ 8.63 (s, 1H), 8.43 (s, 1H), 8.05 (s, 1H),8.03 (s, 2H), 7.86 (d, J=8.5 Hz, 1H), 7.32 (m, 6H), 6.97 (d, J=8.3 Hz,1H), 5.77 (s, 2H), 5.09 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.0, 156.7, 146.1, 140.5, 137.5, 137.3,134.3, 131.0 (q, J_(c-f)=32.8 Hz), 128.9, 128.8, 128.3, 128.2, 126.9,123.6 (q, J_(c-f)=273.4 Hz), 122.4, 121.9, 118.5, 113.3, 110.5, 96.8,95.6, 70.2, 49.2.

(E)-3-(7-(Benzyloxy)-1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL059)

¹H NMR (500 MHz, DMSO-d₆) δ 8.61 (br. s, 1H), 8.45 (br. s, 1H), 7.97(br. s, 1H), 7.60 (br. s, 2H), 7.51 (br. s, 1H), 7.25 (br. s, 2H), 7.16(br. s, 2H), 6.91 (br. s, 1H), 5.94 (s, 2H), 5.13 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.8, 146.6, 145.7, 142.3, 136.6, 135.4,130.7 (q, J_(c-f)=32.8 Hz), 130.5, 128.8, 128.4, 128.0, 127.5, 125.7,124.1, 123.6 (q, J_(c-f)=273.7 Hz), 121.8, 118.3, 111.8, 110.3, 107.1,96.2, 70.3, 52.4.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-methoxy-1H-indol-3-yl)-2-cyanoacrylicacid (JXL060)

¹H NMR (500 MHz, DMSO-d₆) δ 8.99 (s, 1H), 8.71 (s, 1H), 8.05 (s, 1H),8.00 (s, 2H), 7.24 (app. s, 2H), 6.82 (d, J=6.0 Hz, 1H), 5.81 (s, 2H),3.92 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.0, 154.8, 148.6, 140.5, 137.8, 133.3,131.0 (q, J_(c-f)=32.8 Hz), 128.9, 125.4, 123.6 (q, J_(c-f)=273.7 Hz),122.5, 118.5, 116.8, 110.5, 105.0, 104.3, 95.3, 56.2, 49.5.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-5-bromo-1H-indol-3-yl)-2-cyanoacrylicacid (JXL061)

¹H NMR (500 MHz, DMSO-d₆) δ 8.75 (s, 1H), 8.49 (s, 1H), 8.25 (s, 1H),8.06 (s, 1H), 8.02 (m, 3H), 7.64 (app. s, 1H), 7.44 (app. s, 1H), 5.82(s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 145.3, 140.3, 135.5, 135.3, 131.1(q, J_(c-f)=32.8 Hz), 129.8, 128.9, 127.0, 124.7, 123.6 (q,J_(c-f)=274.0 Hz), 122.5, 122.3, 118.5, 113.9, 109.9, 97.7, 49.5.

E-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-6-bromo-1H-indol-3-yl)-2-cyanoacrylicacid (JXL062)

¹H NMR (500 MHz, DMSO-d₆) δ 8.72 (s, 1H), 8.47 (s, 1H), 8.06 (s, 2H),8.03 (s, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.39 (d, J=7.9 Hz, 1H), 5.81 (s,2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 145.6, 140.2, 137.4, 135.3, 131.1(q, J_(c-f)=32.8 Hz), 129.1, 127.0, 125.9, 123.6 (q, J_(c-f)=274.0 Hz),122.5, 121.5, 118.2, 117.2, 114.8, 110.3, 96.9, 49.3.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-6-chloro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL063)

¹H NMR (500 MHz, DMSO-d₆) δ 8.75 (s, 1H), 8.47 (s, 1H), 8.05 (m, 3H),8.00 (d, J=7.4 Hz, 1H), 7.89 (s, 1H), 7.28 (d, J=6.5 Hz, 1H), 5.81 (s,2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 145.6, 140.2, 137.0, 135.4, 131.1(q, J_(c-f)=32.8 Hz), 129.2, 129.0, 126.7, 123.6 (q, J_(c-f)=274.0 Hz),123.3, 122.5, 121.2, 118.2, 111.9, 110.3, 97.0, 49.3.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-7-chloro-1H-indol-3-yl)-2-cyanoacrylicacid (JXL064)

¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.50 (s, 1H), 8.03 (s, 1H),7.97 (d, J=7.5 Hz, 1H), 7.73 (s, 2H), 7.32 (d, J=7.1 Hz, 1H), 7.25 (m,1H), 6.14 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 145.1, 142.1, 136.9, 131.5, 131.2(q, J_(c-f)=32.8 Hz), 127.6, 126.1, 124.1, 123.6 (q, J_(c-f)=274.0 Hz),122.5, 122.0, 118.8, 118.0, 116.9, 110.3, 97.9, 51.8.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-4-bromo-1H-indol-3-yl)-2-cyanoacrylate(JXL065)

¹H NMR (500 MHz, DMSO-d₆) δ 9.67 (s, 1H), 8.71 (s, 1H), 7.86 (s, 1H),7.56 (s, 2H), 7.52 (d, J=7.4 Hz, 1H), 7.17 (m, 2H), 5.55 (s, 2H), 4.37(q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 163.4, 146.9, 137.6, 137.4, 134.3, 132.7(q, J_(c-f)=33.9 Hz), 128.2, 126.7, 125.6, 125.1, 122.8 (q,J_(c-f)=273.4 Hz), 122.7, 118.0, 114.9, 111.8, 109.9, 96.3, 62.2, 50.6,14.3.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-4-fluoro-1H-indol-3-yl)-2-cyanoacrylate(JXL066)

¹H NMR (500 MHz, DMSO-d₆) δ 8.86 (s, 1H), 8.61 (s, 1H), 7.86 (s, 1H),7.56 (s, 2H), 7.23 (m, 1H), 7.01 (m, 2H), 5.54 (s, 2H), 4.37 (q, J=7.1Hz, 2H), 1.39 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 163.1, 157.4 (d, J=243.9 Hz), 147.4, 138.2(d, J=10.1 Hz), 137.8, 133.2, 132.7 (q, J_(c-f)=33.9 Hz), 126.7, 125.3,122.8 (q, J_(c-f)=273.4 Hz), 122.7, 117.9, 116.9 (d, J_(c-f)=17.6 Hz),110.0, 109.0 (d, J_(c-f)=19.5 Hz), 106.6, 97.1, 62.2, 50.7, 14.3.

tert-Butyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylate(JXL068)

¹H NMR (500 MHz, CDCl₃) δ 8.57 (s, 1H), 8.54 (s, 1H), 7.86 (d, J=7.6 Hz,1H), 7.84 (s, 1H), 7.56 (s, 2H), 7.33 (m, 2H), 7.22 (d, J=7.4 Hz, 1H),5.55 (s, 2H), 1.59 (s, 9H).

¹³C NMR (126 MHz, CDCl₃) δ 162.4, 144.7, 138.2, 135.9, 133.0, 132.7 (q,J_(c-f)=32.8 Hz), 128.6, 126.7, 124.5, 123.0, 122.9 (q, J_(c-f)=277.2Hz), 122.6, 119.0, 118.2, 111.1, 110.4, 97.7, 82.9, 50.4, 28.1.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylicacid (JXL069)

¹H NMR (500 MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.47 (m, 3H), 8.09 (s, 2H),8.04 (s, 1H), 7.35 (dd, J=7.1, 4.6 Hz, 1H), 5.84 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 185.9, 164.6, 147.8, 146.1, 145.5, 141.0,135.2, 131.0 (q, J_(c-f)=32.8 Hz), 129.4, 126.9, 123.6 (q, J_(c-f)=274.0Hz), 122.3, 120.1, 119.1, 118.2, 97.1, 47.8.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)-2-cyanoacrylicacid (JXL072)

¹H NMR (500 MHz, DMSO-d₆) δ 8.94 (s, 1H), 8.68 (s, 1H), 8.54 (s, 1H),8.11 (m, 4H), 7.35 (s, 1H), 5.87 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 145.9, 144.2, 140.1, 135.9, 131.1(q, J_(c-f)=32.8 Hz), 129.6, 129.2, 124.7, 123.6 (q, J_(c-f)=274.0 Hz),122.5, 120.0, 119.5, 117.9, 110.1, 97.2, 49.8.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-cyanoacrylicacid (JXL073)

¹H NMR (500 MHz, DMSO-d₆) δ 9.14 (s, 1H), 8.96 (s, 1H), 8.55 (s, 1H),8.40 (d, J=5.1 Hz, 1H), 8.19 (s, 2H), 8.11 (app. s, 2H), 5.95 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.5, 145.5, 140.7, 139.9, 138.1, 134.5,133.6, 131.1 (q, J_(c-f)=32.8 Hz), 129.4, 127.2, 123.6 (q, J_(c-f)=274.0Hz), 122.7, 118.0, 114.6, 109.7, 97.9, 49.8.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylicacid (JXL076)

¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (s, 1H), 8.98 (s, 1H), 8.36 (s, 1H),8.12 (s, 2H), 8.04 (br. s, 1H), 7.47 (br. s, 1H), 5.85 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.4, 148.7, 145.8, 145.7, 140.2, 136.1,135.3, 131.0 (q, J_(c-f)=32.8 Hz), 129.5, 123.6 (q, J_(c-f)=274.0 Hz),122.4, 120.4, 117.7, 116.8, 108.2, 98.1, 48.2.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylicacid (JXL077)

¹H NMR (500 MHz, DMSO-d₆) δ 9.17 (s, 1H), 9.02 (s, 1H), 8.27 (d, J=5.0Hz, 1H), 8.12 (s, 2H), 8.05 (s, 1H), 7.63 (d, J=5.0 Hz, 1H), 5.85 (s,2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.4, 148.2, 145.5, 145.3, 140.2, 135.4,131.0 (q, J_(c-f)=32.8 Hz), 129.6, 124.9, 123.7, 123.6 (q, J_(c-f)=274.0Hz), 122.4, 118.4, 117.7, 108.6, 97.9, 48.2.

Methyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylate(JXL078)

¹H NMR (500 MHz, CDCl₃) δ 8.62 (s, 1H), 8.61 (s, 1H), 7.89 (d, J=7.7 Hz,1H), 7.84 (s, 1H), 7.57 (s, 2H), 7.35 (m, 2H), 7.24 (m, 1H), 5.55 (s,2H), 3.92 (s, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 164.5, 145.8, 138.1, 136.0, 133.5, 132.7 (q,J_(c-f)=32.8 Hz), 128.6, 126.8, 124.7, 123.9, 123.6 (q, J_(c-f)=274.0Hz), 123.2, 119.1, 118.0, 111.2, 110.5, 95.4, 53.0, 50.5.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-(morpholine-4-carbonyl)acrylonitrile(JXL081)

¹H NMR (500 MHz, CDCl₃) δ 8.48 (s, 1H), 8.34 (s, 1H), 7.84 (s, 2H), 7.56(app. s, 2H), 7.32 (m, 2H), 7.22 (m, 1H), 5.54 (s, 2H), 3.77 (br. s,8H).

¹³C NMR (126 MHz, CDCl₃) δ 164.0, 145.2, 138.4, 135.8, 132.7 (q,J_(c-f)=34.0 Hz), 131.8, 128.4, 126.8, 125.0, 124.5, 123.1 (q,J_(c-f)=273.3 Hz), 122.8, 121.8, 119.0, 118.6, 111.4, 110.3, 98.1, 66.7,50.3, 50.0.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylate(JXL082)

¹H NMR (500 MHz, CDCl₃) δ 8.64 (s, 1H), 8.51 (s, 1H), 8.48 (dd, J=4.6,1.2 Hz, 1H), 8.21 (dd, J=7.9, 1.2 Hz, 1H), 7.83 (s, 1H), 7.77 (s, 2H),7.34 (dd, J=7.9, 4.6 Hz, 1H), 5.69 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40(t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.3, 147.6, 145.7, 145.0, 138.5, 132.7,132.3 (q, J_(c-f)=33.6 Hz), 127.9, 127.7, 123.1 (q, J_(c-f)=273.3 Hz),122.5, 120.3, 119.0, 117.6, 109.4, 97.0, 62.3, 48.3, 14.3.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-4-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylate(JXL087)

¹H NMR (500 MHz, CDCl₃) δ 9.22 (s, 1H), 8.77 (s, 1H), 8.32 (d, J=5.2 Hz,1H), 7.83 (s, 1H), 7.77 (s, 2H), 7.31 (d, J=5.2 Hz, 1H), 5.67 (s, 2H),4.37 (q, J=7.1 Hz, 2H), 1.39 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.0, 148.5, 146.2, 145.4, 138.1, 137.4,133.1, 132.4 (q, J_(c-f)=33.7 Hz), 128.0, 122.9 (q, J_(c-f)=273.4 Hz),122.6, 121.8, 117.6, 117.2, 109.7, 97.8, 62.4, 48.6, 14.3.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-4-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyanoacrylate(JXL088)

¹H NMR (500 MHz, CDCl₃) δ 9.40 (s, 1H), 8.78 (s, 1H), 8.22 (d, J=5.1 Hz,1H), 7.83 (s, 1H), 7.77 (s, 2H), 7.49 (d, J=5.1 Hz, 1H), 5.67 (s, 2H),4.37 (q, J=7.1 Hz, 2H), 1.39 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.1, 148.1, 145.7, 145.1, 138.1, 137.4,133.4, 132.4 (q, J_(c-f)=33.7 Hz), 123.7, 122.9 (q, J_(c-f)=273.4 Hz),122.6, 119.7, 117.6, 110.0, 101.4, 97.4, 62.4, 48.6, 14.3.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-5-chloro-1H-indol-3-yl)-2-cyanoacrylate(JXL089)

¹H NMR (500 MHz, CDCl₃) δ 8.60 (s, 1H), 8.50 (s, 1H), 7.85 (s, 1H), 7.83(s, 1H), 7.55 (s, 2H), 7.28 (dd, J=8.7, 1.7 Hz, 1H), 7.14 (d, J=8.7 Hz,1H), 5.54 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.2, 144.8, 137.7, 134.3, 134.1, 132.8 (q,J_(c-f)=33.7 Hz), 129.7, 129.3, 126.7, 125.1, 122.8 (q, J_(c-f)=273.4Hz), 122.7, 118.9, 117.7, 111.6, 110.6, 96.9, 62.3, 50.6, 14.3.

Ethyl(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-4-cyano-1H-indol-3-yl)-2-cyanoacrylate(JXL090)

¹H NMR (500 MHz, CDCl₃) δ 9.29 (s, 1H), 8.78 (s, 1H), 7.87 (s, 1H), 7.68(dd, J=7.4, 0.8 Hz, 1H), 7.56 (s, 2H), 7.49 (dd, J=8.4, 0.8 Hz, 1H),7.38 (dd, J=8.4, 7.4 Hz, 1H), 5.62 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 1.40(t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 162.6, 144.3, 137.3, 136.2, 135.1, 132.9 (q,J_(c-f)=33.7 Hz), 129.4, 127.3, 126.7, 124.1, 123.0, 122.8 (q,J_(c-f)=273.4 Hz), 117.9, 117.5, 115.4, 110.7, 103.5, 98.6, 62.4, 50.6,14.3.

Methyl(E)-1-(3,5-bis(trifluoromethyl)benzyl)-3-(2-cyano-3-ethoxy-3-oxoprop-1-en-1-yl)-1H-indole-4-carboxylate(JXL091)

¹H NMR (500 MHz, CDCl₃) δ 9.36 (s, 1H), 8.71 (s, 1H), 7.93 (dd, 1H,J=7.4, 1.1 Hz, 1H), 7.85 (s, 1H), 7.55 (s, 2H), 7.40 (dd, J=8.3, 1.1 Hz,1H), 7.34 (dd, J=8.3, 7.4 Hz, 1H), 5.58 (s, 2H), 4.37 (q, J=7.1 Hz, 2H),4.04 (s, 3H), 1.40 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 167.8, 163.5, 150.1, 137.8, 137.2, 134.9,132.8 (q, J_(c-f)=33.7 Hz), 126.7, 125.8, 125.1, 123.6, 122.8 (q,J_(c-f)=273.4 Hz), 122.7, 121.7, 118.0, 114.6, 111.3, 96.4, 62.1, 52.6,50.5, 14.3.

Experimental Detail for the Synthesis of JXL024

To the solution of 1-phenyl-1H-indole-3-carbaldehyde (0.4 mmol, 90 mg)in AcOH (3 mL) were added thiazolidine-2,4-dione (1 equiv, 0.4 mmol,46.8 mg) and NaOAc (3 equiv, 98 mg). The reaction mixture was stirred atreflux for 24 hours. After it was cooled to 21° C., the reaction mixturewas filtered by vacuum filtration and washed by AcOH (3 mL×3) and water(5 mL×3). After drying by vacuum, the desired product was produced.yield: 34%, 44 mg.

(Z)-5-((1-Phenyl-1H-indol-3-yl)methylene)thiazolidine-2,4-dione (JXL024)

¹H NMR (500 MHz DMSO-d₆) δ 7.98 (m, 2H), 7.79 (s, 1H), 7.66 (app. d,J=7.7 Hz, 2H), 7.62 (app. t, J=7.7 Hz, 2H), 7.54 (d, J=8.0 Hz, 1H), 7.49(t, J=7.1 Hz, 1H), 7.30 (m, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 172.5, 169.5, 138.4, 136.2, 130.5, 129.9,128.3, 128.2, 125.0, 124.6, 122.4, 121.5, 121.4, 119.6, 113.0, 111.5.

The following compounds were synthesized by a route similar to thatdescribed for JXL024: JXL067, JXL070, JXL072, JXL074, JXL075.

(Z)-2-Imino-5-((1-phenyl-1H-indol-3-yl)methylene)thiazolidin-4-one(JXL023)

¹H NMR (500 MHz, DMSO-d₆) δ 11.94 (br. s, 1H), 9.33 (br. s, 1H), 8.97(s, 1H), 7.64 (m, 10H).

¹³C NMR (126 MHz, DMSO-d₆) δ 180.9, 174.8, 172.5, 138.6, 136.2, 130.5,129.3, 128.1, 126.3, 124.8, 124.5, 122.2, 120.3, 119.7, 113.5, 111.5.

(Z)-5-((1-(3,5-Bis(trifluoromethyl)benzyl)-1H-indol-3-yl)methylene)-2-iminothiazolidin-4-one(JXL067)

¹H NMR (500 MHz, DMSO-d₆) δ 9.21 (s, 1H), 9.01 (s, 1H), 8.03 (s, 1H),7.94 (br. s, 3H), 7.86 (d, J=7.7 Hz, 1H), 7.81 (s, 1H), 7.58 (d, J=7.9Hz, 1H), 7.24 (m, 1H), 7.19 (m, 1H), 5.76 (s, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 180.0, 174.9, 141.4, 136.5, 131.0 (q,J_(c-f)=32.8 Hz), 130.4, 128.5, 127.9, 125.1, 123.8, 123.6 (q,J_(c-f)=274.0 Hz), 122.1, 121.7, 120.8, 119.3, 111.8, 111.3, 48.9.

(Z)-5-((4-Fluoro-1-phenyl-1H-indol-3-yl)methylene)thiazolidine-2,4-dione(JXL070)

¹H NMR (500 MHz, DMSO-d₆) δ 12.45 (br. s, 1H), 8.11 (s, 1H), 7.83 (s,1H), 7.65 (m, 4H), 7.52 (s, 1H), 7.32 (m, 2H), 7.12 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.0, 167.7, 156.9 (d, J_(c-f)=245.7 Hz),138.7 (d, J_(c-f)=10.1 Hz), 137.9, 130.8, 130.5, 128.8, 125.5, 125.4,124.3, 120.0, 116.2 (d, J_(c-f)=18.9 Hz), 110.7, 108.5, 108.1 (d,J_(c-f)=18.9 Hz).

(Z)-5-((6-Fluoro-1-phenyl-1H-indol-3-yl)methylene)thiazolidine-2,4-dione(JXL071)

¹H NMR (500 MHz, DMSO-d₆) δ 12.42 (s, 1H), 8.06 (app. s, 2H), 7.85 (s,1H), 7.67 (m, 2H), 7.62 (m, 2H), 7.50 (m, 1H), 7.31 (d, J=9.6 Hz, 1H),7.16 (t, J=8.5 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.0, 167.7, 160.6 (d, J_(c-f)=245.7 Hz),138.0, 136.3 (d, J_(c-f)=12.6 Hz), 131.0, 130.6, 128.5, 125.0, 124.8,123.5, 121.3 (d, J_(c-f)=10.0 Hz), 120.0, 112.6, 111.1 (d, J_(c-f)=18.9Hz), 98.2 (d, J_(c-f)=18.9 Hz).

(Z)-5-Benzylidenethiazolidine-2,4-dione (JXL074)

¹H NMR (500 MHz, DMSO-d₆) δ 12.60 (br. s, 1H), 7.77 (s, 1H), 7.58 (app.d, J=7.3 Hz, 2H), 7.51 (app. t, J=7.4 Hz, 2H), 7.46 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.4, 167.8, 133.5, 132.3, 130.9, 130.5,129.8, 124.0.

(Z)-5-((1H-Indol-3-yl)methylene)thiazolidine-2,4-dione (JXL075)

¹H NMR (500 MHz, DMSO-d₆) δ 12.28 (s, 1H), 12.11 (s, 1H), 8.03 (s, 1H),7.87 (d, J=7.3 Hz, 1H), 7.72 (s, 1H), 7.48 (d, J=7.6 Hz, 1H), 7.23 (m,1H), 7.18 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.2, 167.8, 136.7, 129.1, 127.3, 125.0,123.5, 121.5, 118.8, 116.7, 112.9, 110.9.

Experimental Detail for the Synthesis of JXL022

To the solution of 4-pyridinecarboxaldehyde (1 mmol, 107 mg) in ethanol(1 mL) were added ethyl 2-cyanoacetate (1.3 equiv, 1.3 mmol, 140 μL) andL-proline (40 mol %, 0.4 mmol, 58 mg). The reaction was stirred at 21°C. for 12 h and yellow solid precipitated gradually. After completion ofthe reaction, ice-cold water (2 mL) was added into the reaction vial.The solid was separated by Buchner funnel filtration and washed withwater (2 mL×3) and dried to afford the desired product, ethyl(E)-2-cyano-3-(pyridin-4-yl)acrylate, which was used for the next stepwithout further purification.

To the solution of (E)-2-cyano-3-(pyridin-4-yl)acrylate (0.21 mmol, 42.4mg) in THF (2 mL) was added 0.5N LiOH solution (3 equiv, 0.4 mmol, 0.8mL). The reaction mixture was stirred at 21° C. for 1 h. After reactioncompletion shown by TLC, THF was evaporated. Concentrated HCl was addeddropwise to acidify the reaction mixture until pH was lower than 1,meanwhile yellow solid precipitated. Ice-cold water (5 mL) was added tothe reaction mixture and the solid was separated by Buchner funnelfiltration and washed with water (5 mL×3). After dried by vacuum, thesolid was washed by 2 mL of solvent mixture (hexanes/EtOAc=5:1) 5 to 10times and monitored by TLC until non-polar impurities disappear.Finally, the purity of the product was checked by NMR. yield: 64%, 23.4mg.

Ethyl (E)-2-cyano-3-(pyridin-4-yl)acrylate (JXL022)

¹H NMR (500 MHz, CDCl₃) δ 8.81 (d, J=5.2 Hz, 2H), 8.18 (s, 1H), 7.74 (d,J=5.2 Hz, 2H), 4.41 (q, J=7.1 Hz, 2H), 1.41 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 161.2, 152.0, 151.0, 137.9, 123.2, 114.2,108.2, 63.2, 14.0.

The following compounds were synthesized by a route similar to thatdescribed for JXL022: JXL030, JXL031, JXL032, JXL033, JXL034, JXL042,JXL43, JXL044, JXL045, JXL046, JXL047, JXL048, JXL049.

(E)-2-Cyano-3-(2-fluorophenyl)acrylic acid (JXL030)

¹H NMR (500 MHz, DMSO-d₆) δ 8.49 (s, 1H), 8.31 (t, J=7.4 Hz, 1H), 7.63(m, 1H), 7.36 (t, J=7.4 Hz, 1H), 7.29 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 162.9, 161.5 (d, J_(c-f)=256.2 Hz), 145.4(d, J_(c-f)=7.8 Hz), 135.0 (d, J_(c-f)=9.2 Hz), 128.7, 124.7, 119.8 (d,J_(c-f)=10.9 Hz), 115.8 (d, J_(c-f)=21.9 Hz), 114.9, 105.9.

(E)-2-Cyano-3-(4-fluorophenyl)acrylic acid (JXL032)

¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (s, 1H), 8.10 (m, 2H), 7.29 (m, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.2 (d, J_(c-f)=255.2 Hz), 163.5, 153.1,133.3 (d, J_(c-f)=9.3 Hz), 128.3, 116.0 (d, J_(c-f)=22.4 Hz), 115.3,103.2.

(E)-2-cyano-3-(3-(trifluoromethyl)phenyl)acrylic acid (JXL033)

¹H NMR (500 MHz, CD₃OD) δ 8.24 (s, 1H), 8.17 (m, 2H), 7.80 (d, J=7.8 Hz,1H), 7.70 (app. t, J=7.8 Hz, 1H).

(E)-2-Cyano-3-(4-(trifluoromethyl)phenyl)acrylic acid (JXL034)

¹H NMR (500 MHz, DMSO-d₆) δ 8.39 (s, 1H), 8.17 (d, J=7.7 Hz, 2H), 7.84(d, J=7.7 Hz, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 162.8, 152.6, 135.4, 133.1 (q, J_(c-f)=32.9Hz), 131.0, 125.7, 123.7 (q, J_(c-f)=272.2 Hz), 114.8, 106.7.

(E)-2-Cyano-3-(3-fluoro-4-methylphenyl)acrylic acid (JXL042)

¹H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.78 (m, 2H), 7.49 (t, J=8.0Hz, 1H), 2.30 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 163.6, 160.9 (d, J_(c-f)=244.6 Hz), 153.5,133.0, 131.6 (d, J_(c-f)=7.5 Hz), 130.8 (d, J_(c-f)=17.6 Hz), 127.2,117.9 (d, J_(c-f)=23.9 Hz), 116.4, 104.5, 15.0.

(E)-2-Cyano-3-(3,4-difluorophenyl)acrylic acid (JXL043)

¹H NMR (500 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.09 (d, J=8.0 Hz, 1H), 7.94(br. s, 1H), 7.67 (d, J=8.8 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 163.3, 152.3, 152.2 (dd, J_(c-f)=255.4,12.6 Hz), 149.9 (dd, J_(c-f)=248.2, 12.6 Hz), 129.8, 128.8, 120.1 (d,J-f=17.6 Hz), 119.1 (d, J_(c-f)=17.6 Hz), 116.3, 105.8.

(E)-2-Cyano-3-(2,4-difluorophenyl)acrylic acid (JXL044)

¹H NMR (500 MHz, DMSO-d₆) δ 14.2 (br. s, 1H), 8.27 (s, 1H), 8.24 (m,1H), 7.52 (m, 1H), 7.35 (dt, J=8.6, 2.2 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 165.5 (dd, J_(c-f)=255.8, 12.6 Hz), 163.1,161.9 (dd, J_(c-f)=270.5, 12.6 Hz), 145.1, 131.1, 117.0, 115.9, 113.5(d, J_(c-f)=22.7 Hz), 106.8, 105.6 (t, J_(c-f)=26.5 Hz).

(E)-3-(3,5-bis(trifluoromethyl)phenyl)-2-cyanoacrylic acid (JXL045)

¹H NMR (500 MHz, CD₃OD) δ 8.59 (s, 2H), 8.48 (s, 1H), 8.19 (s, 1H).

(E)-3-(2-Chloro-3-(trifluoromethyl)phenyl)-2-cyanoacrylic acid (JXL046)

¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (s, 1H), 8.23 (d, J=7.7 Hz, 1H), 8.05(d, J=7.4 Hz, 1H), 7.75 (app. t, J=7.9 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 162.6, 150.5, 134.4, 133.5, 132.1, 131.2,128.9, 128.3 (q, J_(c-f)=30.2 Hz), 123.1 (q, J_(c-f)=274.1 Hz) 115.1,111.1.

(E)-2-Cyano-3-(4-fluoro-3-(trifluoromethyl)phenyl)acrylic acid (JXL047)

¹H NMR (500 MHz, DMSO-d₆) δ 8.46 (dd, J=7.1, 1.8 Hz, 1H), 8.44 (s, 1H),8.40 (m, 1H), 7.75 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 163.3, 161.1 (d, J_(c-f)=262.1 Hz), 152.2,137.6 (d, J_(c-f)=10.1 Hz), 130.5, 129.3, 122.6 (q, J_(c-f)=272.8 Hz),118.9 (d, J_(c-f)=21.2 Hz), 118.2 (qd, J_(c-f)=32.9, 12.6 Hz), 116.2,106.3.

(E)-2-Cyano-3-phenylacrylic acid (JXL048)

¹H NMR (500 MHz, DMSO-d₆) δ 8.33 (s, 1H), 8.02 (m, 2H), 7.59 (m, 3H).

¹³C NMR (I 26 MHz, DMSO-d₆) δ 163.7, 154.9, 133.6, 132.0, 131.1, 129.8,116.5, 104.3.

(E)-2-cyano-3-(4-hydroxyphenyl)acrylic acid (JXL049)

¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (s, 1H), 7.95 (d, J=8.8 Hz, 2H), 6.92(d, J=8.8 Hz, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.2, 163.0, 153.7, 133.6, 122.8, 117.2,116.3, 99.2.

Experimental Detail for the Synthesis of JXL079

A flask containing Mg powder (10 mmol, 240 mg) and a stir bar was sealedand vacuumed and refilled with argon three times. Anhydrous diethylether (32 mL) and bis(trifluoromethyl)benzyl bromide (8 mmol, 1.46 mL)was added to the reaction flask. The reaction mixture was stirred andrefluxed for 30 min and then ground dry ice powder (5 g) was added intothe reaction flask. After 1 h, the reaction was complete as shown byTLC. The extra Mg powder was filtered off and the solvent was evaporatedunder vacuum. 1N HCl (20 mL) was added to the residue and theprecipitate was filtered and dried to provide the desired carboxylicacid.

A flask containing a stir bar was sealed, vacuumed and refilled withargon three times. Anhydrous dichloromethane (20 mL) and DIBAL (1 M inhexanes, 6 mmol, 6 mL) were added to the flask. The crude carboxylicacid (2 mmol, 544 mg) dissolved in dry dichloromethane (10 mL) was addedto the reaction flask at −78° C. After 2 h, the reaction was complete asshown by TLC and it was then quenched by adding sat. ammonium chloride(10 mL). The resulting mixture was extracted with dichloromethane (20mL×3) and the organic phases were combined and evaporated on therotavap. The residue was purified by flash column chromatography(hexanes:ethyl acetate=10:1) to afford the desired product2-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol (yield: 90%, 464 mg).

To a solution of 2-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol (0.2 mmol,51.6 mg) in dichloro-methane (2 mL) was added triethylamine (0.22 mmol,31 μL) and mesyl chloride (MsCl, 0.2 mmol, 17 μL) at 0° C. Afterstirring for 1 h, the reaction was complete as shown by TLC. The solventwas removed by flowing air over the open flask. The residue was purifiedby flash column chromatography (hexanes:ethyl acetate=10:1) to affordthe desired product 3,5-bis(trifluoromethyl)phenethyl methanesulfonate(yield: 82%, 55.1 mg).

A flask containing NaH (60%, 0.22 mmol, 8.8 mg) and a stir bar wassealed, vacuumed and refilled with argon three times. Anhydrous THF (3mL) and a solution of tert-butyl (E)-2-cyano-3-(1H-indol-3-yl)acrylate(0.2 mmol, 53.6 mg) in THF (2 mL) were added into the reaction flask.The reaction mixture was stirred for 30 min and then3,5-bis(trifluoromethyl)phenethyl methanesulfonate (0.164 mmol, 55.1 mg)in 2 mL THF was added. The reaction was stirred for 24 h and quenched bysat. NH₄Cl solution. The resulting mixture was extracted withdichloromethane (4 mL×3) and the organic phases were combined andevaporated on the rotavap. The residue was purified by flash columnchromatography (hexanes:ethyl acetate=10:1) to afford the desiredproduct tert-butyl(E)-3-(1-(3,5-bis(trifluoromethyl)phenethyl)-1H-indol-3-yl)-2-cyanoacrylate(yield: 68%, 69 mg).

To a solution of methyl tert-butyl(E)-3-(1-(3,5-bis(trifluoromethyl)phenethyl)-1H-indol-3-yl)-2-cyanoacrylate(0.1 mmol, 50.8 mg) in dichloromethane (2 mL) was added trifluoroaceticacid (3 equiv, 0.3 mmol, 34 μL). The reaction mixture was stirred at 21°C. for 30 min and a yellow solid precipitated. After the reaction wascomplete as shown by TLC, the reaction solvent was evaporated by flowingair over the open flask. The solid was washed by 2 mL of solvent mixture(hexanes/EtOAc=5:1) 5 to 10 times and monitored by TLC until all thenon-polar impurities disappeared. Finally, the purity of the product waschecked by NMR. yield: 87%, 39 mg.

(E)-3-(1-(3,5-Bis(trifluoromethyl)phenethyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL079)

¹H NMR (500 MHz, CD₃OD) δ 8.51 (s, 1H), 8.14 (s, 1H), 7.83 (d, J=7.3 Hz,1H), 7.74 (s, 1H), 7.52 (m, 3H), 7.31 (m, 2H), 4.64 (t, J=6.4 Hz, 2H),3.35 (t, J=6.3 Hz, 2H).

¹³C NMR (126 MHz, CD₃OD) δ 165.3, 145.3, 141.1, 136.1, 133.6, 131.3 (q,J_(c-f)=32.8 Hz), 129.3, 128.3, 123.7, 123.3 (q, J_(c-f)=272.5 Hz),122.3, 120.3, 118.1, 117.6, 110.6, 109.6, 94.1, 47.8, 35.0.

Experimental Detail for the Synthesis of JXL080

To a solution of ethyl (E)-2-cyano-3-(1H-indol-3-yl)acrylate (0.5 mmol,112 mg) in dichloromethane (5 mL) were added2-(3,5-bis(trifluoromethyl)phenyl)acetic acid (0.55 mmol, 150 mg), DMAP(catalytic amount, 6 mg), and DCC (0.5 mmol, 103 mg) at 0° C. Themixture was allowed to reach 21° C. and stirred overnight. The whiteprecipitate was filtered, and the resulting solution was concentrated invacuum. The solid was purified by flash column chromatography(hexanes:ethyl acetate=10:1) to afford the desired product (yield: 78%,192.6 mg).

Ethyl(E)-3-(1-(2-(3,5-bis(trifluoromethyl)phenyl)acetyl)-1H-indol-3-yl)-2-cyanoacrylate(JXL080)

¹H NMR (500 MHz, CDCl₃) δ 8.90 (s, 1H), 8.52 (s, 1H), 8.49 (d, J=8.1 Hz,1H), 7.88 (s, 1H), 7.86 (s, 2H), 7.78 (d, J=7.5 Hz, 1H), 7.47 (m, 2H),4.50 (s, 2H), 4.41 (q, J=7.1 Hz, 2H), 1.43 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 168.0, 162.2, 144.2, 135.6, 134.4, 132.3 (q,J_(c-f)=33.6 Hz), 130.1, 129.8, 128.8, 127.8, 127.3, 125.5, 123.1 (q,J_(c-f)=273.3 Hz), 118.3, 117.1, 117.0, 115.9, 101.8, 62.8, 41.9, 14.3.

Experimental Detail for the Synthesis of JXL083

To a solution of1-(3,5-bis(trifluoromethyl)benzyl)-1H-indole-3-carbaldehyde (10 mmol,3.71 g) in acetone (60 mL) was added 2-methyl-2-butene (9 mL), NaH₂PO₄(3 equiv, 4.4 g) and NaClO₂ (6.6 mmol, 6 g) in 6 mL water. The reactionmixture was stirred at 21° C. for 24 h. After the reaction was completeas shown by TLC, the reaction solvent was evaporated on the rotavap. Thecrude material was dissolved in ethyl acetate (30 mL) and water (30 mL)and extracted with ethyl acetate (30 mL×3). The organic phase wascombined, dried with sodium sulfate and evaporated on the rotavap. Thesolid was purified by flash column chromatography (hexanes:ethylacetate=2:1) to afford the desired product1-(3,5-bis(trifluoromethyl)benzyl)-1H-indole-3-carboxylic acid (yield:89%, 3.44 g).

A 100 mL round bottom flask with a stir bar containing the carboxylicacid (5 mmol, 1935 mg) from the previous step was sealed, vacuumed andrefilled with argon three times. To the flask was added 50 mLdichloromethane and oxalyl chloride (25 mmol, 2.1 mL) dropwise. Thereaction mixture was stirred at 21° C. for 1.5 h. The reaction solventwas evaporated by vacuum and the resulting compound was used for thenext step.

A 100 mL round bottom flask with a stir bar was sealed, vacuumed andrefilled with argon three times. Diisopropylamine (5.5 mmol, 765 μL) andTHF (10 mL) were added into the flask and it was cooled to −78° C. nBuLi(2.5 M in hexanes, 5 mmol, 2 mL) was added slowly to the flask. Afterthe mixture had stirred for 30 min, a solution of ethyl 2-cyanoacetate(5 mmol, 590 μL) in THF (10 mL) was added slowly to the flask. After themixture had stirred for 1 h, a solution of the acyl chloride (5 mmolfrom previous step) in THF (5 mL) was added slowly to the reactionmixture. After 1 h, the reaction was quenched by adding aqueous 1M HClsolution (10 mL) and extracted with ethyl acetate (10 mL×3). The organicphase was combined, dried with sodium sulfate and evaporated by rotavap.The solid was purified by flash column chromatography (hexanes:ethylacetate=10:1) to afford the desired product Ethyl(Z)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyano-3-hydroxyacrylate(JXL083) (yield: 80%, 1.93 g).

¹H NMR (500 MHz, CDCl₃) δ 14.61 (s, 1H), 8.67 (s, 1H), 8.31 (dd, J=7.01.3 Hz, 1H), 7.84 (s, 1H), 7.57 (s, 2H), 7.33 (m, 2H), 7.21 (d, J=7.4Hz, 1H), 5.53 (s, 2H), 4.40 (q, J=7.1 Hz, 2H), 1.41 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 179.1, 172.2, 138.1, 136.0, 135.7, 132.7 (q,J_(c-f)=33.6 Hz), 126.9, 124.5, 123.7, 123.6, 122.6, 122.9 (q,J_(c-f)=273.4 Hz), 121.8, 118.3, 110.1, 109.6, 73.1, 62.3, 50.4, 14.3.

Experimental Detail for the Synthesis of JXL084

To a solution of JXL083 (0.5 mmol, 240 mg) in dichloromethane (10 mL)was added pyridine (0.5 mmol, 40 μL) and acetyl chloride (1.0 mmol, 84μL). The reaction mixture was stirred for 1 h and TLC indicated that thereaction was complete. The reaction solvent was evaporated by flowingair over the open flask. The residue was purified by flash columnchromatography (hexanes:ethyl acetate=10:1) to afford the desiredproduct Ethyl(Z)-3-acetoxy-3-(1-(3,5-bis(trifluoro-methyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylate(JXL084) (yield: 86%, 225 mg).

¹H NMR (500 MHz, CDCl₃) δ 8.56 (s, 1H), 7.90 (m, 1H), 7.85 (s, 1H), 7.61(s, 2H), 7.32 (m, 2H), 7.24 (m, 1H), 5.51 (s, 2H), 4.29 (q, J=7.1 Hz,2H), 2.48 (s, 3H), 1.36 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 166.9, 161.4, 137.7, 136.2, 135.5, 132.8 (q,J_(c-f)=33.9 Hz), 127.0, 126.7, 124.6, 123.6, 122.8 (q, J_(c-f)=273.4Hz), 122.7, 121.8, 117.7, 110.7, 109.8, 89.3, 61.8, 50.5, 29.7, 21.3,14.2.

Experimental Detail for the Synthesis of JXL085

To a solution of JXL083 (0.5 mmol, 240 mg) in dichloromethane 10 mL wasadded triethylamine (1.0 mmol, 139.5 μL) and phosphoryl chloride (0.55mmol, 520 μL). The reaction mixture was stirred for 1 h at reflux andTLC indicated that the reaction was complete. The reaction solvent wasevaporated by flowing air over the open flask. The residue was purifiedby flash column chromatography (hexanes:ethyl acetate=10:1) to affordthe desired product Ethyl(Z)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-3-chloro-2-cyanoacrylate(JXL085) (yield: 84%, 210 mg).

¹H NMR (500 MHz, CDCl₃) δ 7.91 (s, 1H), 7.85 (s, 1H), 7.77 (m, 1H), 7.65(s, 2H), 7.63 (s, 1H), 7.30 (m, 2H), 5.48 (s, 2H), 4.18 (q, J=7.1 Hz,2H), 1.18 (t, J=7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 161.0, 155.6, 138.1, 136.2, 135.4, 133.4,132.7 (q, J_(c-f)=33.7 Hz), 127.1, 126.6, 124.5, 124.3, 122.8 (q,J_(c-f)=273.4 Hz), 121.5, 115.8, 122.1, 110.4, 102.5, 62.4, 50.0, 13.9.

Experimental Detail for the Synthesis of JXL086

Diethyl(E)-(2-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-1-cyanovinyl)phosphonate(JXL086)

To the solution of1-(3,5-bis(trifluoromethyl)benzyl)-1H-indole-3-carboxaldehyde (1 mmol,371 mg) in ethanol (3 mL) were added diethyl cyanomethylphosphate (1.3equiv, 1.3 mmol, 204 μL) and L-proline (40 mol %, 0.4 mmol, 58 mg). Thereaction was stirred at 50° C. for 24 h. After completion of thereaction as indicated by TLC, the reaction solvent was evaporated byflowing air over the open flask. The solid was purified by flash columnchromatography (hexanes:ethyl acetate=2:1) to afford the desired productJXL086 (yield: 90%, 477 mg).

¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H), 8.33 (d, J=19.7 Hz, 1H), 7.86(d, J=7.9 Hz, 1H), 7.83 (s, 1H), 7.58 (s, 2H), 7.31 (m, 2H), 7.22 (m,1H), 5.54 (s, 2H), 4.21 (m, 4H), 1.39 (t, J=7.0 Hz, 6H).

¹³C NMR (126 MHz, CDCl₃) δ 149.8, 138.3, 135.8, 132.7 (q, J_(c-f)=33.7Hz), 132.6, 128.2, 126.8, 124.5, 123.0, 122.9 (q, J_(c-f)=273.4 Hz),122.5, 119.0, 117.8 (d, J_(c-p)=11.3 Hz), 112.2 (d, J_(c-p)=18.9 Hz),110.3, 91.6 (d, J_(c-p)=207.9 Hz), 63.2, 50.4, 16.3.

JXL095 was Synthesized by the Similar Route as JXL086.

Diethyl(E)-(2-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1-cyanovinyl)phosphonate(JXL095)

¹H NMR (500 MHz, CDCl₃) δ 8.58 (s, 1H), 8.46 (dd, J=4.7, 1.4 Hz, 1H),8.24 (d, J=19.5 Hz 1H), 8.20 (dd, J=8.0, 1.4 Hz, 1H), 7.82 (s, 1H), 7.77(s, 2H), 7.32 (dd, J=8.0, 4.7 Hz, 1H), 5.67 (s, 2H), 4.22 (m, 4H), 1.40(t, J=7.1 Hz, 6H).

¹³C NMR (126 MHz, CDCl₃) δ 149.2 (d, J_(c-p)=8.2 Hz), 147.4, 145.6,138.6, 132.4 (q, J_(c-f)=33.7 Hz), 132.0, 127.9, 127.8, 123.0 (q,J_(c-f)=273.4 Hz), 122.4, 119.9, 118.9, 117.4 (d, J_(c-p)=11.3 Hz),110.5 (d, J_(c-p)=19.5 Hz), 92.8 (d, J_(c-p)=205.1 Hz), 63.4, 48.3,16.3.

Experimental Detail for the Synthesis of JXL096

(E)-(2-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-1-cyanovinyl)phosphonicacid (JXL096)

A solution of JXL086 (30 mg, 0.057 mmol) in dichloromethane (2 mL) wascooled to 0° C. and bromotrimethylsilane (40 μL, 0.3 mmol) was addeddropwise under argon. The mixture was warmed to 21° C. and stirred for12 h. The solvent was evaporated under vacuum and the resulting residuewas then dissolved in methanol (2 mL). The mixture was stirred at 21° C.for 2 h. Evaporation of all volatiles under vacuum gave the phosphoricacid JXL096 (yield: 92%, 25 mg).

¹H NMR (500 MHz, CD₃OD) δ 8.58 (s, 1H), 8.25 (d, J=19.6 Hz, 1H), 7.90(s, 1H), 7.87 (m, 1H), 7.76 (s, 2H), 7.45 (m, 1H), 7.31 (m, 2H), 5.75(s, 2H).

¹³C NMR (126 MHz, CD₃OD) δ 146.7 (J_(c-p)=7.2 Hz), 140.2, 136.1, 132.1,131.9 (q, J_(c-f)=33.7 Hz), 128.0, 127.2, 123.8, 122.3, 123.2 (q,J_(c-f)=273.4 Hz), 121.4, 118.2, 117.4 (d, J_(c-p)=11.3 Hz), 111.5(J_(c-p)=18.4 Hz), 110.5, 94.6 (d, J_(c-p)=201.2 Hz), 49.1.

Experimental Detail for the Synthesis of JXL092

To a solution of methyl1-(3,5-bis(trifluoromethyl)benzyl)-3-formyl-1H-indole-4-carboxylate (1mmol, 429 mg) in ethanol (3 mL) were added tert-butyl 2-cyanoacetate(1.3 equiv, 1.3 mmol, 183 μL) and L-proline (40 mol %, 0.4 mmol, 58 mg).The reaction was stirred at 21° C. for 12 h and a yellow solidprecipitated gradually. After completion of the reaction, ice-cold water(2 mL) was added into the reaction. The solid was separated by Buchnerfunnel filtration and washed with water (2 mL×3) and dried to afford thedesired product. yield: 95%, 524 mg.

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-4-(methoxycarbonyl)-1H-indol-3-yl)-2-cyanoacrylicacid (JXL092)

To the solution of methyl(E)-1-(3,5-bis(trifluoromethyl)benzyl)-3-(3-(tert-butoxy)-2-cyano-3-oxoprop-1-en-1-yl)-1H-indole-4-carboxylate(0.5 mmol, 276 mg) in dichloromethane (2 mL) was added trifluoroaceticacid (3 equiv, 1.5 mmol, 0.2 mL). The reaction mixture was stirred at21° C. for 30 min and a yellow solid precipitated. After the reactionwas complete as shown by TLC, the reaction solvent was evaporated byflowing air over the open flask. The solid was washed by 2 mL of solventmixture (hexanes/EtOAc=5:1) 5 to 10 times and monitored by TLC until allthe non-polar impurities disappeared. Finally, the purity of the productwas checked by NMR. yield: 90%, 223 mg.

¹H NMR (500 MHz, CDCl₃) δ 9.09 (s, 1H), 8.58 (s, 1H), 7.75 (d, J=7.5 Hz,1H), 7.68 (s, 1H), 7.47 (s, 2H), 7.34 (d, J=8.2 Hz, 1H), 7.20 (app. t,J=7.9 Hz, 1H), 5.51 (s, 2H), 3.87 (s, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 167.8, 165.1, 149.5, 138.2, 137.2, 134.8,132.3 (q, J_(c-f)=33.7 Hz), 126.8, 126.2, 125.5, 124.8, 123.3, 122.8 (q,J_(c-f)=273.4 Hz), 122.3 118.2, 114.7, 110.9, 97.2, 52.4, 50.2.

Experimental Detail for the Synthesis of JXL094

(E)-3-(1-(3,5-Bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylamide(JXL094)

The mixture of(E)-3-(1-(3,5-bis(trifluoromethyl)benzyl)-1H-indol-3-yl)-2-cyanoacrylicacid JXL001 (0.1 mmol, 43.8 mg) and thionyl chloride (0.5 ml) wasrefluxed for 1 h. After concentration under vacuum, the resulting acylchloride was dissolved in 1 ml benzene and 30% ammonia in water (1 ml)was added. The reaction mixture was stirred at 21° C. for 24 h. Aftercompletion of the reaction as indicated by TLC, the reaction solvent wasevaporated under vacuum. The solid was purified by flash columnchromatography (hexanes:ethyl acetate=2:1) to afford the desired productJXL094 (yield: 85%, 37 mg).

1H NMR (500 MHz, CDCl₃) δ 8.70 (s, 1H), 8.47 (s, 1H), 7.92 (d, J=7.1 Hz,1H), 7.85 (s, 1H), 7.58 (s, 2H), 7.34 (m, 2H), 7.23 (m, 1H), 5.55 (s,2H).

¹³C NMR (126 MHz, CDCl₃) δ 163.0, 144.8, 138.1, 136.0, 132.7 (q,J_(c-f)=33.7 Hz), 132.5, 128.6, 126.8, 126.1, 124.6, 123.1, 122.9 (q,J_(c-f)=273.4 Hz), 122.6, 119.3, 111.3, 110.3, 96.1, 50.4.

Additional exemplary compounds of the present invention can be preparedby methods analogous to those described above.

Example 2: Treatment of Epithelial Cells with Exemplary Compounds

To determine whether these compounds could promote cellular lactateproduction, we treated cultured epithelial cells with the compounds andmeasured lactate levels in the culture media using a Nova BiomedicalBioProfile Basic Analyzer. Briefly, cultured epithelial cells weretreated with DMSO, UK-5099 (also called JXL001), or certain of theexemplary compounds disclosed herein for 24-30 hours, and media lactatelevels were measured and normalized to cell number and duration of theexperiment to acquire a cellular lactate production rate (nmol lactate,million cells, hour).

Lactate production rates of treated cells are shown in FIGS. 8, 9, and12. As expected based on the present disclosure, since they are UK-5099analogues, most of the novel compounds assayed increased lactateproduction. Furthermore, the total cell count following treatment withthe UK-5099 analogues is shown in FIG. 13. Most of the compounds weretolerated by the cells. A separate assay was performed to calculate theEC50 of some of the compounds as shown in FIG. 10.

Example 3: In Vivo Test of Exemplary Compounds

To determine the efficacy of the compounds on the hair cycle, mice wereshaved at postnatal day 50, and topically treated every other day with acompound disclosed herein suspended in lotion in every other day for 2weeks, and pictures were taken. As seen in FIG. 11, all the analoguesthat showed the ability to promote lactate production in the in vitroassay were also able to stimulate hair growth over the course of 2weeks.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. A compound of formula I or a pharmaceutically acceptable saltthereof,

wherein: Y is carboxyl, ester, amide, or

R¹ is H, aryl, aralkyl, or aralkylacyl, and is optionally substituted byone or more R⁵; R² is CN or carboxyl; R³ is CN or carboxyl; R⁴ isindependently alkyl, alkenyl, alkynyl, azido, halo, hydroxy, carboxyl,ester, or CN; R⁵ is independently selected from alkyl, alkoxy, or halo;R¹⁰ is H or alkyl; and n is 0-4; provided that the compound is not


2. (canceled)
 3. The compound of claim 1, wherein Y is


4. The compound of claim 1, wherein R¹⁰ is H.
 5. (canceled)
 6. Thecompound of claim 1, wherein Y is ester or carboxyl.
 7. The compound ofclaim 1, wherein R² is CN.
 8. The compound of claim 1, wherein R² iscarboxyl.
 9. The compound of claim 1, wherein R¹ is H.
 10. The compoundof claim 1, wherein R¹ is aralkyl.
 11. The compound of claim 1, whereinR¹ is aralkyl and is substituted by one or more R⁵.
 12. The compound ofclaim 1, wherein R¹ is aralkylacyl and is substituted by one or more R⁵.13. The compound of claim 1, wherein R¹ is substituted by one or two R⁵,and wherein each R⁵ is independently selected from fluoroalkyl orfluoro.
 14. The compound of claim 1, wherein R¹ is substituted by twoR⁵, and wherein each R⁵ is trifluoromethyl.
 15. The compound of claim 1,wherein R⁴ is selected from iodo, fluoro, alkenyl, CN, azido, alkynyl,fluoroalkyl, carboxyl, and ester.
 16. The compound of claim 1, whereinR⁴ is not chloro or bromo.
 17. The compound of claim 1, wherein thecompound is represented by formula Ia:

wherein R⁶ is H, alkyl, aryl, or aralkyl,
 18. A compound selected from:

or a pharmaceutically acceptable salt thereof.
 19. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable excipient.
 20. (canceled)
 21. (canceled)
 22. A method ofenhancing lactate production in a cell, comprising contacting the cellwith a compound of claim
 1. 23. (canceled)
 24. A method of promotinghair growth, comprising administering to a patient a compound ofclaim
 1. 25. A method of treating a condition or disorder affecting hairgrowth, comprising administering to a patient a compound of claim 1.26-32. (canceled)